A new livebearing fish of the genus Limia (Cyprinodontiformes: Poeciliidae) from Lake Miragoane, Haiti

Abstract

Limia islai, a new species of livebearing fish, is described from Lake Miragoane in south‐western Haiti on Hispaniola. The new species has a conspicuous barred pattern consisting of several (4–12) black bars along the body, ray 4p serrae of the gonopodium in males with 10 segments and origin of dorsal fin in females slightly behind the origin of the anal fin. Although the new species colour pattern is similar to that of the humpbacked limia Limia nigrofasciata Regan 1913, L. islai sp. nov. has exclusive morphological features, such as slender body, lack of hump anterior to dorsal fin in males and presence of specific features in the gonopodial suspensory, which allow an unambiguous diagnosis from L. nigrofasciata. L. islai further differs from L. nigrofasciata in reproductive behaviour since L. islai males rely on sneak copulations and gonopodial thrusting, whereas L. nigrofasciata display an elaborate courtship behaviour. The new species is also genetically distinct in both nuclear (Rh, Myh6) and mitochondrial (12S, ND2, D‐loop, Cytb) genes from other species in the genus showing reciprocal monophyly. The description of this new Limia species from Lake Miragoane confirms this lake as an important centre of endemism for the genus, with a total of eight endemic species described so far.

Full text

REGULAR PAPER
A new livebearing fish of the genus Limia (Cyprinodontiformes:
Poeciliidae) from Lake Miragoane, Haiti
Rodet Rodriguez-Silva
1
| Pablo F. Weaver
2
1
Department of Biology, University of
Oklahoma, Norman, OK
2
Department of Biology, University of La
Verne, La Verne, CA
Correspondence
Rodet Rodriguez-Silva, Department of Biology,
University of Oklahoma, 730 Van Vleet Oval,
Norman, OK 73019, USA.
Email: rodet.rodriguez.silva-1@ou.edu
Funding information
Financial support was given by a University of
La Verne Faculty Research Grant, a University
of Colorado Boulder Museum Research Grant,
and a University of Colorado Boulder
Department of Ecology and Evolutionary
Biology departmental graduate student grant.
The Caribaea Initiative and the National
Geographic Society (WW-054R-17) provided
additional funding for this research.
Abstract
Limia islai, a new species of livebearing fish, is described from Lake Miragoane in
south-western Haiti on Hispaniola. The new species has a conspicuous barred pat-
tern consisting of several (4–12) black bars along the body, ray 4p serrae of the
gonopodium in males with 10 segments and origin of dorsal fin in females slightly
behind the origin of the anal fin. Although the new species colour pattern is similar to
that of the humpbacked limia Limia nigrofasciata Regan 1913, L. islai sp. nov. has
exclusive morphological features, such as slender body, lack of hump anterior to dor-
sal fin in males and presence of specific features in the gonopodial suspensory, which
allow an unambiguous diagnosis from L. nigrofasciata.L. islai further differs from
L. nigrofasciata in reproductive behaviour since L. islai males rely on sneak copulations
and gonopodial thrusting, whereas L. nigrofasciata display an elaborate courtship
behaviour. The new species is also genetically distinct in both nuclear (Rh, Myh6) and
mitochondrial (12S, ND2, D-loop, Cytb) genes from other species in the genus show-
ing reciprocal monophyly. The description of this new Limia species from Lake
Miragoane confirms this lake as an important centre of endemism for the genus, with
a total of eight endemic species described so far.
KEYWORDS
endemism, gonopodium, Lake Miragoane, Limia, morphology
1|INTRODUCTION
The genus Limia Poey 1854 includes small livebearing fishes endemic
to several Caribbean islands. Specifically, Hispaniola is considered to
be a radiation centre for this genus since 17 species are currently rec-
ognized as valid from this island whereas only one endemic species is
reported from Cuba, Jamaica and Grand Cayman (Burgess & Franz,
1989; Chambers, 1987; Rodriguez, 1997; Weaver et al., 2016a).
Although Limia is considered a freshwater fish genus, some species
are tolerant to varying salinity levels and may even be found in hyper-
saline coastal lagoons (Haney & Walsh, 2003; Weaver et al., 2016b).
However, most species occur in inland waters, including species
locally distributed in mountain streams.
Recent phylogenetic analyses have provided consistent results
suggesting a South American origin of this genus via dispersal and/or
vicariance from northern South America (Hrbek et al., 2007; Palacios
et al., 2016; Reznick et al., 2017; Weaver et al., 2016a). Lake
Miragoane, located in the southern part of Haiti, may be considered
an important area of origin and diversification of Limia species since
seven endemic species are currently known from that lake. This spe-
cies flock is represented by six species described and included by
Rivas (1980) in the subgenus Odontolimia Rivas, 1980: Limia grossidens
Rivas, 1980, L. fuscomaculata Rivas, 1980, L. ornata Regan 1913,
L. garnieri Rivas, 1980, L. immaculata Rivas, 1980, and L. miragoanensis
Rivas, 1980) and one species of the subgenus Limia:L. nigrofasciata
Regan 1913.
In this work we describe a new species of Limia from specimens
recently collected at the north bight of Lake Miragoane in Haiti. We
examined morphological, molecular and some behavioural features to
formally describe this new livebearing fish that is somewhat common
Received: 16 December 2019 Accepted: 25 February 2020
DOI: 10.1111/jfb.14301
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1360 © 2020 The Fisheries Society of the British Isles J Fish Biol. 2020;96:1360–1369.wileyonlinelibrary.com/journal/jfb

in the specialized aquarium trade under the name “Tiger Limia”but
has never been properly evaluated for species identity.
2|MATERIALS AND METHODS
The care and use of experimental animals complied with the Univer-
sity of Oklahoma animal welfare laws, guidelines and policies as
approved by Animal Welfare Assurance on file with the Office of Lab-
oratory Animal Welfare under the assurance number D16-00153
(A3240-01). Fish were collected as part of a survey of the native
freshwater fish fauna of the Lake Miragoane in Haiti. Fish were eutha-
nized in the field using MS-222 and then preserved in 75% ethanol.
Surgical procedures were performed in previously euthanized individ-
uals (four specimens total) to remove scales and guts for clearing and
staining of bones.
2.1 |Morphological analysis
Morphological data were collected from wild-caught specimens col-
lected in June 2019 in Lake Miragoane, Haiti (Figure 1). Fish were
collected as part of a survey of the native freshwater fish fauna of
the lake. A total of 33 specimens (19 males and 14 females) were
euthanized using MS-222 and then preserved in 75% ethanol. All
specimens were examined under an Olympus SZX7 (Olympus Cor-
poration, Tokyo, Japan) stereomicroscope with direct and transmit-
ted light and magnification 50×. Characters used by Rivas (1980) in
the description of five Limia species from Lake Miragoane in Haiti
were measured in all specimens using the methods described in
Rivas (1978). Two additional gonopodial characters, segments distal
to ray 3 keel and ray 4 grooved segments, were included in the spe-
cies description (Chambers, 1987). Scale counts were done
according to Miller (1948). Morphometric measurements were taken
using a DigitalAid caliper (to the nearest 0.1 mm) following Rivas
(1963) and Rivas and Fink (1970). The length of the caudal peduncle
was measured from the middle of the dorsal fin to the beginning of
the caudal fin. Proportional body and fin measurements were
avoided in the species description since these characters can be
affected by several environmental factors (Hubbs & Springer, 1957;
Rivas, 1963; Rivas & Fink, 1970). Two males and two females were
cleared and single stained using the protocol of Taylor and van Dyke
(1985) for description of internal support of gonopodium and
gonopodium tip. In addition, X-ray images were taken for five males
and three females. The nomenclature used for osteological features
of internal support of the gonopodium followed Rodriguez (1997).
2.2 |Molecular analysis
A sample of 20 specimens of L. islai sp. of an aquarium stock resulting
from a source population collected in 2001 by Dominic Isla in Lake
Miragoane (Figure 1) was used in the molecular analysis. Comparative
groups of other Limia species from the Dominican Republic were col-
lected in the field (Table 1). In addition, the Haitian species
L. nigrofasciata, also from Lake Miragoane, the Jamaican species
L. melanogaster (Gunther 1866), the Cuban species L. vittata
(Guichenot 1853) and the Grand Cayman species L. caymanensis
Rivas & Fink, 1970 were obtained through aquarium stocks
maintained at the University of Colorado. Unfortunately, the other
FIGURE 1 Map showing the collection localities for specimens included in this study. Enlargement shows greater detail of Lake Miragoane in
Haiti, where number 1 is the source locality of aquarium specimens and number 2 is the locality for wild caught specimens (holotype and
paratypes)
RODRIGUEZ-SILVA AND WEAVER 1361
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Limia species from Lake Miragoane, including the entire Odontolimia
subgenus, were unavailable for this analysis. All specimens used in the
molecular analyses were preserved in 80% ethanol prior to DNA
extraction and kept as vouchers at the University of Colorado
Museum.
Genomic DNA was extracted from ethanol-preserved caudal
peduncle muscle tissue following the manufacturer's protocol for the
DNeasy Kit (Qiagen Inc., Valencia, CA, USA). Six loci, including four
mitochondrial (12S, ND2, D-loop, Cytb) and two nuclear (Rh, Myh6),
were examined for each of the 45 individuals included in the phylog-
eny. All genes were amplified via PCR using the primer sequences
listed in Table 2. DNA sequences were edited with Geneious Pro
5.6.6 and alignments were performed with Clustal W (Thompson
et al., 1997). The resulting gene sequences are available in GenBank,
accession numbers MT028461–MT028480.
Phylogenetic analyses were performed using both Bayesian and
maximum likelihood analyses. Trees were rooted with related out-
groups Pamphoricthys hollandi (Henn), Poecilia latipunctata Meek
1904, P. reticulata Peters 1859, and P. caucana (Steindachner 1880)
obtained through GenBank, as well as P. hispaniolana Rivas, 1978,
P. dominicensis (Evermann and Clark 1906) and P. elegans (Trewavas
1948) obtained from Weaver (2005). Gene sequences for outgroups
were obtained through GenBank (KX023907–KX024245), with addi-
tional sequences listed in Supporting Information Table S1. The pri-
mary goal of the phylogenetic analysis was to evaluate genetic
distinctness and reciprocal monophyly among L. islai sp. nov. and
closely related groups. Preliminary trees on a combined mitochondrial
dataset (12S, ND2, Cytb, D-loop) and a combined nuclear dataset (Rh,
MYH6) were constructed to evaluate species level relationships. The
topologies of the mitochondrial and nuclear trees were consistent
TABLE 1 Locality data and taxon codes for outgroups used in this study
Species name Locality Taxon code
Limia perugiae Macasias 1847058.8100N, 7130029.3000 WPM
Rio San Juan 1848026.3500N, 711407.5500W PRSJ
Arroyo Salado 1757019.3000N, 7111016.7300 W PAS
Laguna Oviedo 1747054.4900N, 7121049.9700 W PLO
Guanarate 1830035.7100N, 7110040.5800 WPG
La Zurza 1823052.2200N, 7134011.8900W PLZ
L. sulphurophila La Zurza 1823052.2200N, 7134011.8900W SLZ
L. sp. Mata de Maiz 184047.1700N, 7115029.9500W LMM
L. sp. Presa Las Damas 1818058.0300N, 7134038.5300W LPLD
L. sp. Pozo Ecologico 1757053.6400N, 7138023.8600 W PPE, LPE
L. sp. Cabo Rojo 1754053.2800N, 7139026.7100 W LCR
L. zonata Rio Yuna 1916012.6500N, 6926046.6400 W ZRY
Rio San Juan 1914050.1700N, 6919050.8100W ZRSJ
L. versicolor Bassima 1842023.1400N, 7011048.4600 WVB
L. versicolor
L. tridens
L. dominicensis
Rio Cana Arriba 1842017.9300N, 702608.2000 W
Genbank, Dominican Republic
Genbank, Haiti
VRCA
L. tridens
L. dominicensis
L. nigrofasciata Aquarium Specimen, Lake Miragoane, Haiti L_nigrofasciata
L. caymanensis Aquarium Specimen, Grand Cayman L_caymanensis
L. vittata Aquarium Specimen, Cuba L_ vittata
L. melanogaster Aquarium Specimen, Jamaica L_ melanogaster
Poecilia hispaniolana La Cienega 194035.5400N, 7049058.6600W HLC
Rio Ocoa 1831041.6300N, 7030021.5600 W HRO
Poecilia dominicensis Arroyon 1844013.3800N, 7011037.9000 W DAR
Jimenoa 19906.9500N, 7038029.8300 W DJI
Poecilia elegans Caño Piedra 19005.7200N, 7026043.9100W ECP
Rio Verde 184607.2100N, 709052.2400 W ERV
Pamphorichthys hollandi Genbank, Brazil Pamphorichthys hollandi
Poecilia latipunctata Genbank, Mexico P_latipunctata
Poecilia reticulata Genbank, South America P_reticulata
Poecilia caucana Genbank, Central and South America P_caucana
1362 RODRIGUEZ-SILVA AND WEAVER
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hence the sequences were combined into a concatenated dataset of
mitochondrial and nuclear sequences. PartitionFinder (Lanfear et al.,
2012) was used to evaluate the appropriate nucleotide substitution
models and partition scheme on combined dataset. PartitionFinder
allowed us to evaluate various partition schemes, including the frag-
ments of 12S ribosomal DNA and D-loop, and each codon position
independently in all of the protein coding genes (ND2, Cytb, Rh, and
MYH6). Bayesian analyses were performed using Mr. Bayes version
3.1.2 (Ronquist & Huelsenbeck, 2003), as implemented in Geneious
Pro 5.6.6. The Marcov chain Monte Carlo (MCMC) simulation was
performed utilizing two independent runs, using eight chains and
10 million generations, sampling every 1000 generations. The first
25% of each run was established as burn-in and discarded. The
remaining trees were used to compute a 50% majority rule consensus
tree with posterior probability (PP) for branch support. Maximum like-
lihood (ML) trees were built using PhyML (Guindon & Gascuel, 2003)
as implemented in Geneious Pro 5.6.6. ML support for nodes was
established with 1000 likelihood bootstrap replicates.
Institutional abbreviations used: OMNH, Sam Noble Museum of
Natural History, University of Oklahoma; USMN, National Museum of
Natural History; MNHNSD, Museo de Historia Natural “Prof. Eugenio
de Jesús Marcano,”Santo Domingo, Dominican Republic.
3|RESULTS
3.1 |L. islai, sp. nov (Figures 2–7)
Holotype: OMNH 86867, adult male 29.5 mm standard length
(SL) (Figure 2). Haiti, Nippes Department, Miragoane, Lake
Miragoane's northern bight, on the roadside of Route Nationale
2. Coordinates 1825035.400N, 7302057.700 W; R. Rodriguez-Silva,
J. Josaphat, P. Torres-Pineda, B. Pierre Michard, 5 June 2019.
Paratypes: Same origin, collection date and collectors as holotype.
OMNH 86868, 1 female, 35 mm SL and 1 male, 33 mm SL; OMNH
86869, 1 female, 29 mm SL and 1 male, 31.5 SL; OMNH 86927,
1 female, 29 mm SL and 1 male, 25.5 mm SL (cleared and stained
specimens). MNHNSD 22.10650, 1 male, 31 mm SL; MNHNSD
22.10651, 1 female, 33.5 mm SL; MNHNSD 22.10652, 1 male,
28.5 mm SL and 1 female, 32 mm SL; MNHNSD 22.10653, 1 male,
28 mm SL (cleared and stained specimen).
TABLE 2 Genes, primer sequences and corresponding sources
used in this study
Gene and primer sequence Source
12S
12Sa CL
50-CTGGGATTAGATACCCCACTA-30
12Sb CL
50-TGAGGAGGGTGACGGGCGGT-30
Meredith et al.
(2010)
ND2
C-TRP-Pam
50-GTCTAAGGAATTATCCTAAG-30
t-MetCT
50-ACCCTGAACATGACSGYTAAAA-30
Hamilton (2001)
D-loop
L15926
50-TCAAAGCTTACACCAGTCTTGTAAACC-30
H16498
50-CCTGAAGTAGGAAC AGATG-30
Shields & Kocher
(1991)
Cyt b
LA
50-GTGACTTGAAAAACCACCGTTG-30
HA
50-CAACGATCTCCGGTTTACAAGAC-30
Schmidt et al.
(1998)
MYH6
AF1 F
50-CATMTTYTCCATCTCAGATAATGC-30
AR1 R
50-ATTCTCACCACCATCCAGTTGAA-30
Li et al. (2007)
Rh
Rh193F F
50-CNTATGAATAYCCTCAGTACTACC-30
Rh1039R R 5
50-TGCTTGTTCATGCAGATGTAGA-30
Chen & Mayden
(2009)
FIGURE 2 L. islai sp. nov., holotype OMNH 86867, adult male
29.5 mm SL
FIGURE 3 Coloration pattern in a live female of L. islai sp. nov.
collected in Lake Miragoane (specimen was not preserved)
RODRIGUEZ-SILVA AND WEAVER 1363
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3.2 |Diagnosis
L. islai sp. nov. can be easily distinguished from all other members of
the genus Limia (sensu lato) except L. nigrofasciata by the noticeable
black bars on both sides of the body of males and females. However,
the barred pattern is frequently more conspicuous in L. islai than in
L. nigrofasciata, with darker and wider vertical bars. L. islai further
differs from L. nigrofasciata and other sympatric species such as
L. grossidens,L. fuscomaculata,L. garnieri,L. immaculata,
L. miragoanensis and L. ornata in having the origin of the dorsal fin of
females slightly posterior to the origin of the anal fin, while in all the
other aforementioned species the origin of the dorsal fin is either
slightly anterior or above the origin of the anal fin. The species also
differs from all other described species in the genus in body shape
since L. islai has a slender body with body depth approximately equal
to the caudal peduncle length. In all other Limia species, body depth is
shorter than caudal peduncle length except in L. nigrofasciata, which
has deeper body in relation to the length of the caudal peduncle (more
conspicuous in males). The number of serrae in the ray 4p of the
gonopodium in males (10 segments in all measured specimens) also
differentiates L. islai from other congeners. The gonapophyses and
uncinated processes that support the gonopodium are thicker and
more developed in L. islai compared to other species such as
L. nigrofasciata (Figures 5a,b and 6).
3.3 |Description
Body compressed and slender with body depth about the same length
of caudal peduncle (Table 3); post-anal region very compressed
towards caudal peduncle. Pre-dorsal profile slightly concave from
snout tip to posterior edge of eye; convex from distal margin of eye
FIGURE 4 Gonopodium tip of a cleared and stained male of
L. islai sp. nov., paratype OMNH 86872, adult male 25.5 mm SL
FIGURE 5 X-ray images of gonopodial suspensory of (a) L. islai sp. nov., adult male 30.5 mm SL, (b) L. nigrofasciata USNM 78245, adult male
40.2 mm SL, (c) L. miragoanensis USNM 220531, adult male, and (d) L. garnieri USNM 220531, adult male. Scale bars represent 1 cm
1364 RODRIGUEZ-SILVA AND WEAVER
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to origin of dorsal fin; slightly concave or straight from dorsal fin origin
to caudal fin. Pre-anal profile convex and post-anal profile slightly
concave or straight (Figures 2 and 3). Short, rounded dorsal fin with
eight rays; distinctive black spot present at base of fourth to eighth
fin rays. Origin of dorsal fin in females slightly posterior of origin of
anal fin. Pectoral-fin rays usually 13. Caudal fin slightly truncate and
symmetrical in both sexes (Figure 3). Gonopodial segments of ray 4p
serrae 10 and usually 16 segments distal to ray 4p serrae (Figure 4
and Table 4). The internal support of gonopodium with two
gonapophyses with uncinated processes, common to all Limia species
(Rodriguez, 1997) (Figure 5). Mouth superiorly oriented; teeth of outer
row in both jaws spatulate (Figure 7). Vertebral column with 28 verte-
brae in all measured individuals.
3.4 |Colour in live specimens
Light olive-green background that turns yellow in the dorsal area, base
of caudal and dorsal fins and snout. Both sexes with 4–12 black bars
on both flanks of body (Figures 2 and 3). Presence of blurred and tiny
black spots on edge of dorsal fin, which are more evident in males.
3.5 |Colour in alcohol
Yellowish to light brown background with 4–12 conspicuous black
bars on both flanks of body. A rounded, distinctive black spot can be
seen at the base of sixth to ninth fin rays. All fins have whitish colora-
tion (Figure 2).
3.6 |Distribution and ecological notes
L. islai sp. nov. is known only from Lake Miragoane in south-western
Haiti. The species was collected in a small, clear water path exiting the
north end of Lake Miragoane and was also observed in the lake itself
(Figure 8). Unfortunately, little else is known about the ecology of this
species but it seems that the species prefers habitats with some
submerged vegetation and muddy bottoms. L. islai was sympatric
with Gambusia beebei,Nandopsis haitiensis,Limia nigrofasciata,
L. garnieri,L. miragoanensis,L. immaculata and some introduced spe-
cies, such as Oreochromis aureus and Tilapia rendali.
3.7 |Etymology
We named this species in honour of one of the first collectors, Domi-
nic Isla, who passed away before the species was ever formally evalu-
ated. The species is also known as “Tiger Limia”due to its distinct
pattern of black vertical stripes.
3.8 |Molecular analysis
The dataset comprising the concatenated mitochondrial and nuclear
data provided strong support for nodes, while still allowing for resolu-
tion among most of the closely related groups (Figure 9). There was
strong support from both the ML and Bayesian analyses (100% and
1.0, respectively) for reciprocal monophyly among L. islai sp. nov. and
its closest sister group L. nigrofasciata. Corrected pairwise genetic dif-
ferences between L. islai. and L. nigrofasciata were 0.3% for 12 s,
0.5–0.8% for cyt b, 0.5–0.9% for ND2 and 0.9% for D-loop. Compara-
tively, the within-species genetic distances for both of these taxa was
nearly zero. Sister to the clade comprising L. islai and L. nigrofasciata is
a large clade of other Hispaniolan taxa, which include the closely
related L. perugiae (Evermann and Clark 1906) like species (L. perugiae,
L. tridens (Hilgendorf 1889), L. sulphurophila Rivas, 1980, and several
unidentified populations from the south-west Dominican Republic).
FIGURE 6 Close-up of the gonopodial suspensorium in a cleared
and stained male of L. islai sp. nov., paratype OMNH 86872, adult
male 25.5 mm SL
FIGURE 7 Mouth and teeth of outer row in both jaws of a
paratype OMNH 86870 of L. islai sp. nov
RODRIGUEZ-SILVA AND WEAVER 1365
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TABLE 3 Morphometric measurements for holotype and paratypes (10 males and 6 females) of L. islai sp. nov
Character Holotype
Males (n= 10) Females (n=6)
Mean ± S.D. Range Mean ± S.D. Range
Standard length 29.50 30.56 ± 2.24 27.50–33.00 32.58 ± 2.04 29.00–35.00
Head length 8.00 8.56 ± 0.32 8.00–9.00 9.00 ± 0.55 8.00–9.50
Eye diameter 3.00 3.06 ± 0.18 3.00–3.50 3.08 ± 0.20 3.00–3.50
Dorsal length (snout to dorsal fin origin) 18.00 18.44 ± 1.24 16.5–20.00 20.67 ± 1.63 17.50–22.00
Pectoral length (snout to pectoral fin origin) 9.50 9.81 ± 0.65 9.00–11.00 10.58 ± 0.86 9.00–11.50
Pelvic length (snout to pelvic fin origin) 12.00 12.38 ± 0.95 11.00–13.00 13.58 ± 1.43 12.00–15.00
Anal length (snout to anal fin origin) 15.50 16.06 ± 0.72 15.00–17.00 19.50 ± 1.70 16.50–21.50
Caudal peduncle depth 5.00 6.13 ± 0.58 5.50–7.00 5.83 ± 0.68 5.50–7.00
Caudal peduncle length 9.00 9.38 ± 0.88 8.00–10.50 9.33 ± 0.52 9.00–10.00
Body depth 9.00 9.43 ± 0.86 8.00–10.50 9.33 ± 0.75 8.50–10.50
Note: The mean value is provided for all measurements in mm.
TABLE 4 Counts for the holotype (indicated by asterisks) and paratypes of L. islai sp. nov
Character Males (n= 10) Females (n=6)
Dorsal rays 8* (10) 8 (6)
Caudal rays 16 (2) 17* (8) 15 (1) 16 (1) 17 (4)
Pectoral rays 12 (2) 13* (8) 12 (2) 13 (3) 14 (1)
Anal rays ––– 7 (6)
Segments distal to ray 3 keel 20* (6) 21 (1) 22 (3) ––––
Ray 4a grooved segments 12 (2) 14 (2) 15* (6) ––––
Ray 4p serrae 10* (10) ––––
Segments distal to ray 4p serrae 14 (2) 15 (1) 16* (7) ––––
Lateral scales 26 (2) 27* (7) 28 (1) 26 (3) 27 (1) 28 (2)
Pre-dorsal scales 12 (4) 13* (6) 12 (2) 13 (3) 14 (1)
Pre-orbital pores 2* (9) 4 (1) 2 (4) 3 (2)
Pre-opercular pores 5* (3) 6 (2) 7 (5) 5 (2) 6 (1) 7 (3)
Note: Values in parentheses indicate number of specimens examined with that count.
FIGURE 8 Habitats where L. islai
sp.nov was collected. Left: south
bight of Lake Miragoane, which
corresponds with locality number 1 in
Figure 1. Right: north end of Lake
Miragoane, which corresponds with
locality number 2 in Figure 1
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Corrected pairwise genetic differences between the larger L. perugiae
like clade and L. islai were 1.3% for 12 s, 0.9–2.3% for cyt b, 2.0–2.2%
for ND2 and 4.3% for D-loop.
4|DISCUSSION
The description of this new poeciliid adds another species record to
the impressive biodiversity of the genus Limia on Hispaniola, especially
in Lake Miragoane on the Haitian side of the island. Lake Miragoane is
one of the few places in the Neotropics that holds a high biodiversity
of endemic livebearing fishes. The lake is isolated in terms of connec-
tivity with other water bodies, since no tributary rivers or streams lead
to the lake. This topography, coupled with the early colonization of
south-western Hispaniola by the Limia lineage (Weaver et al., 2016a),
may explain the radiation of Limia species in the lake. The geographic
area deserves more scientific work in light of the serious conservation
challenges facing Haiti's biodiversity (Hedges et al., 2018).
The description of L. islai sp. nov. solves a nomenclatural gap that
has been present for over 18 years since the species became very
L. perugiae like species (Hispaniola)
(includes L. perugiae, L. tridens, and L. sulphurophila)
PG1
PM1
PM3
L_tridens
LPE5
PRSJ1
PAS1
PPE2
SLZ1
SLZ2
LMM4
LPLD3
PLO1
LCR1
TL5
TL7
TL1
TL2
TL4
L_nigrofasciata2
L_nigrofasciata3
L_nigrofasciata4
L_nigrofasciata1
L_dominicensis
L_viata
L_caymanensis
VB2
VRCA1
ZRSJ3
ZRY1
L_melanogaster1
L_melanogaster2
DAR4
DJi15
HLC5
HRO2
ECP2
ERV4
P_lapunctata
1
100
1
100 1
100
1
100
1
100
1
100
1
100
1
100
1
100
1
100
1
100
1
100
1
100
1
100
1
100
1
100
1
89
1
89
1
99
1
100
0.9
95
0.9
98
P_reculata
P_caucana
0.03
PLZ3
L. islai n. sp. (Lake Miragoane, Hispaniola)
L. nigrofasciata (Lake Miragoane, Hispaniola)
L. dominicensis (Hispaniola)
L. caymanensis (Grand Cayman)
L. viata (Cuba)
L. versicolor (Hispaniola)
L. zonata (Hispaniola)
L. melanogaster (Jamaica)
Poecilia dominicensis (Hispaniola)
Poecilia hispaniolana (Hispaniola)
Poecilia elegans (Hispaniola)
Pamphorichthys hollandi (South America)
Poecilia spp. (Central and South America)
FIGURE 9 ML phylogeny, based on concatenated dataset of mitochondrial (12 s, ND2, cytb, D-loop) and nuclear (Rh, MYH6) sequences
showing the arrangement of L. islai sp. nov. with other members of the genus Limia. Numbers above and below nodes indicate Bayesian posterior
probability and ML support. The geographic range for taxa is also indicated in parentheses. The clade labelled as L. perugiae like species was
unresolved with incomplete lineage sorting and included representatives of L. perugiae,L. tridens and L. sulphurophila
RODRIGUEZ-SILVA AND WEAVER 1367
FISH

popular among aquarists. For several years the species herein
described has been indistinctly referred to as “Tiger Limia”and was
frequently confounded with L. nigrofasciata, maybe due the similar
coloration pattern and body shape of juveniles of L. nigrofasciata.
However, there are several morphological and behavioural features
that distinguish L. islai as a different, valid species. L. islai males have
slender bodies and they lack courtship behaviour, apparently relying
exclusively on sneak copulations and gonopodial thrusting (Weaver,
2015), whereas L. nigrofasciata males are humpbacked and display a
large, ornamented dorsal fin along with elaborate sigmoid, tail wag
and lateral displays to attract females (Cruz & Munger, 1999;
Farr, 1984).
Another morphological feature that distinguishes L. islai and
L. nigrofasciata as different species is the structure of the gonopodial
suspensory (Figures 5 and 6). L. islai has well-developed and robust
gonapophyses with uncinated processes as part of the internal sup-
port of the gonopodium while L. nigrofasciata has fine gonapophyses.
The development of the osteological support of the gonopodium in
L. islai could be associated with its reproductive behaviour based on
gonopodial thrusting that requires sudden movements of this struc-
ture for copulation. These slight variations of the gonopodial suspen-
sory present in L. islai might represent effective reproductive barriers
between these two closely related species.
L. islai is also genetically distinct (in both nuclear and mitochon-
drial genes) from other species in the genus. Furthermore, it demon-
strates reciprocal monophyly among closely related species, indicating
substantial lack of gene flow and complete lineage sorting. The new
species is certainly not, as some aquarists have suggested, a hybrid
between L. perugiae and L. nigrofasciata since it can be identified as a
valid species by molecular analysis. Although average corrected
pairwise genetic distances to its sister taxon, L. nigrofasciata, may
seem low (0.3% for 12 s, 0.5–0.8% for cyt b, 0.5–0.9% for ND2 and
0.9% for D-loop) when compared to species level differences found in
other poeciliids (Bagley et al., 2015), they represent reciprocally
monophyletic groups and a fair representation of species-level differ-
ences comparable or greater than genetic distances observed among
other well-established species, e.g., L. perugiae from L. tridens and
L. sulphurophila (Figure 9). The average pairwise differences between
L. islai and L. nigrofasciata are greater than the divergence levels found
in cichlid species flocks (0.1–0.25% difference) that have seen very
recent diversification (Malinsky et al., 2018). The close relationship
between L. islai and L. nigrofasciata only gives evidence to a recent
diversification event, kept separate by morphological and behavioural
reproductive isolation.
4.1 |Comparative material
Limia nigrofasciata: BMNH 1913.7.25.15 (1 ex. Syntype), Limia
nigrofasciata: BMNH 1913.3.6.21 (1 ex. Syntype), Limia grossidens:
USMN 220523 (1 ex. Holotype); Limia fuscomaculata: USMN 220525
(1 ex. Holotype), USMN 220526 (1 ex. Paratype); Limia garnieri:
USMN 220527 (1 ex. Holotype); Limia immaculata: USMN 220529
(1 ex. Holotype), USMN 220530 (1 ex. Paratype); Limia miragoanensis:
USMN 220531 (1 ex. Holotype); Limia pauciradiata: USMN 220533
(1 ex. Holotype); Limia yaguajali: USMN 220535 (1 ex. Holotype);
Limia sulphurophila: USMN 220537 (2 exs. Paratypes); Limia rivasi:
USMN 232484 (2 exs. Paratypes); Limia nigrofasciata: USMN 78243
(3 exs.), USMN 78245 (4 exs.); Limia versicolor: USMN 88338 (5 exs.);
Limia ornata: USMN 164769 (2 exs.); Limia zonata: USMN 89002
(2 exs.); Limia melanogaster: USMN 102200 (3 exs.); Limia dominicensis:
USMN 120359-60 (2 exs.), USMN 132111 (4 exs.); Limia perugiae:
USMN 382409 (3 exs.); Limia vittata: USMN 167692 (4 exs.), USMN
331925 (3 exs.); Limia tridens: USMN 305527 (1 ex.); Limia
melanonotata USMN 305523 (3 exs.), USMN 122629 (2 exs.).
ACKNOWLEDGEMENTS
We would like to give special thanks to Alexander Cruz and Greg
Sage for providing samples for analysis, as well as the countless
undergraduates in the Cruz and Weaver laboratories that have stud-
ied the Tiger Limia over the years. Thanks also to our colleagues at
the University of Oklahoma, University of La Verne and the Univer-
sity of Colorado for your insight and guidance. Thanks to Vanessa
Morales, Andrew Martin, Robert Guralnick, David Stock, Dena Smith,
Frank Cezilly and Ingo Schlupp for assistance with fieldwork, analyses
and reading drafts. Thanks to Lynne Parenti and Sandra Raredon for
the support provided to analyse comparative material at the NMNH.
Thanks to James Maclaine, Simon P. Loader, Emanuell Ribeiro,
Ricardo Betancur and Dahiana Arcila for pictures provided and feed-
back of manuscript. Thanks also to our Dominican and Haitian col-
leagues for assistance in the field, including Arlen Marmolejo, Miguel
Landestoy, Patricia Torres Pineda, Marcos Rodriguez, Carlos
Rodriguez, Carlos Suriel, James Josaphat and Pierre Michard Beaujour,
as well as the corresponding ministries in both countries for permits.
This study was funded by the Caribaea Initiative, the National Geo-
graphic Society (WW-054R-17), a University of La Verne Faculty
Research Grant, a University of Colorado Boulder Museum Research
Grant, and a University of Colorado Boulder Department of Ecology
and Evolutionary Biology graduate student grant.
ORCID
Rodet Rodriguez-Silva https://orcid.org/0000-0002-7463-8272
REFERENCES
Bagley, J. C., Alda, F., Breitman, M. F., Bermingham, E., Berghe, E. P., &
Johnson, J. B. (2015). Assessing species boundaries using multilocus
species delimitation in a morphologically conserved group of neotropi-
cal freshwater fishes, the Poecilia sphenops species complex
(Poeciliidae). PLoS One,10(4), 1–30.
Burgess, G. H., & Franz, R. (1989). Zoogeography of the Antillean freshwa-
ter fish fauna. In C. A. Woods & F. E. Sergile (Eds.), Biogeography of the
West Indies: Patterns and perspectives (pp. 263–304). Gainesville, FL:
Sandhill Crane Press.
Chambers, J. (1987). The cyprinodontiform gonopodium, with atlas of the
gonopodia of the fihshes of the genus Limia.Journal of Fish Biology,30,
389–418.
Chen, W. J., & Mayden, R. L. (2009). Molecular systematics of the
Cyprinoidea (Teleostei: Cypriniformes), the world's largest clade of
1368 RODRIGUEZ-SILVA AND WEAVER
FISH

freshwater fishes: further evidence from six nuclear genes. Molecular
Phylogenetics and Evolution,52, 544–549.
Cruz, A., & Munger, L. (1999). The biology of the humpback limia (Limia
nigrofasciata). Livebearers: Bulletin of the American Livebearer Associa-
tion,158,6–12.
Farr, J. A. (1984). Premating behavior in the subgenus Limia (pices:
poeciliidae): sexual selection and the evolution of courtship. Zeitschrift
für Tierpsychologie,65, 152–165.
Guindon, S., & Gascuel, O. (2003). A simple, fast, and accurate algorithm to
estimate large phylogenies by maximum likelihood. Systematic Biology,
52, 696–704.
Hamilton, A. (2001). Phylogeny of Limia (Teleostei: Poeciliidae) based on
NADH dehydrogenase subunit 2 sequences. Molecular Phylogenetics
and Evolution,19, 277–289.
Haney, D. C., & Walsh, S. J. (2003). Influence of salinity and temperature
on the physiology of Limia melanonotata (Cyprinodontiformes:
Poeciliidae): A search for abiotic factors limiting insular distribution in
Hispaniola. Caribbean Journal of Science,39(3), 327–337.
Hedges, B. S., Cohen, W. B., Timyan, J., & Yang, Z. (2018). Haiti's biodiver-
sity threatened by nearly complete loss of primary forest. Proceedings
of the National Academy of Sciences of the United States of America,11,
850–11855.
Hrbek, T., Seckinger, J., & Meyer, A. (2007). Molecular phylogeny of the
Poeciliidae (Teleostei, Cyprinodontiformes): biogeographic and life-
history implications. Molecular Phylogenetics abd Evolution,43,986–998.
Hubbs, C., & Springer, V. G. (1957). A revision of the Gambusia nobilis spe-
cies group, with descriptions of three new species, and notes on their
variation, ecology and evolution. Texas Journal of Sciences,9, 279–327.
Lanfear, R., Calcott, B., Ho, S. Y., & Guindon, S. (2012). PartitionFinder: com-
bined selection of partitioning schemes and substitution models for
phylogenetic analyses. Molecular Biology and Evolution,29,1695–1701.
Li, C., Ortí, G., Zhang, G., & Lu, G. (2007). A practical approach to phy-
logenomics: the phylogeny of ray-finned fish (Actinopterygii) as a case
study. BMC Evolutionary Biology,7, 44.
Malinsky, M., Svardal, H., Tyers, A. M., Miska, E. A., Genner, M. J.,
Turner, G. F., & Durbin, R. (2018). Whole-genome sequences of
Malawi cichlids reveal multiple radiations interconnected by gene flow.
Nature, Ecology and Evolution,2, 1940–1955.
Meredith, R. W., Pires, M. N., Reznick, D. N., & Springer, M. S. (2010).
Molecular phylogenetic relationships and the evolution of the placenta
in Poecilia (Micropoecilia) (Poeciliidae: Cyprinodontiformes). Molecular
Phylogenetics and Evolution,55, 631–639.
Miller, R. R. (1948). The cyprinodont fishes of the Death Valley system of
eastern California and southwestern Nevada. Miscellaneous Publica-
tions Museum of Zoology, University of Michigan,68,7–155.
Palacios, M., Voelker, G., Rodriguez, L. A., Mateos, M., & Tobler, M. (2016).
Phylogenetic analyses of the subgenus Mollienesia (Poecilia, Poeciliidae,
Teleostei) reveal taxonomic inconsistencies, cryptic biodiversity, and
spatio-temporal aspects of diversification in middle America. Molecular
Phylogenetics and Evolution,103, 230–244.
Reznick, D. N., Furness, A. I., Meredith, R. W., & Springer, M. S. (2017).
The origin and biogeographic diversification of the fishes in the family
Poeciliidae. PLoS One,12,1–20.
Rivas, L. R. (1963). Subgenera and species groups in the poeciliid fish
genus Gambusia Poey. Copeia,1963, 331–347.
Rivas, L. R. (1978). A new species of poeciliid fish of the genus Poecilia
from Hispaniola, with reinstatement and redescription of
P. dominicensis (Evermann and Clark). Northeast Gulf Science,2,
98–112.
Rivas, L. R. (1980). Eight new species of poeciliid fishes of the genus Limia
from Hispaniola. Northeast Gulf Science,4,28–38.
Rivas, L. R., & Fink, W. L. (1970). A new species of poeciliid fish of the
genus Limia from the Island of Grand Cayman, B.W.I. Copeia,2,
270–274.
Rodriguez, C. M. (1997). Phylogenetic analysis of the tribe Poeciliini
(Cyprinodontiformes: Poeciliidae). Copeia,4, 663–679.
Ronquist, F., & Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian phyloge-
netic inference under mixedmodels. Bioinformatics,19, 1572–1574.
Schmidt, T. R., Bielawski, J. P., & Gold, J. R. (1998). Molecular phyloge-
netics and evolution of the cytochrome b gene in the cyprinid genus
Lythrurus (Actinopterygii: Cypriniformes). Copeia,1998,14–22.
Shields, G. L., & Kocher, T. D. (1991). Phylogenetic relationships of North
American Ursids based on analysis of mitochondrial DNA. Evolution,
45, 218–221.
Taylor, W. R., & van Dyke, G. C. (1985). Revised procedures for staining
and clearing small fishes and other vertebrates for bone and cartilage
study. Cybium,9, 107–119.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., &
Higgins, D. G. (1997). The CLUSTAL X windows interface: flexible
strategies for multiple sequence alignment aided by quality analyses
tools. Nucleic Acid Research,25, 4876–4882.
Weaver, P.F. (2005) Inferring patterns of West Indian colonization and speci-
ation through a molecular phylogenetic analysis of Poecilia (Masters the-
sis). University of Colorado Boulder, Boulder, CO.
Weaver, P.F (2015). Colonizing the Caribbean: Biogeography,physiology,and
diversification of Limia (Poeciliidae) (Doctoral thesis, University of Colo-
rado Boulder). Retrieved from https://scholar.colorado.edu/ebio_
gradetds/69
Weaver, P. F., Cruz, A., Johnson, S., Dupin, J., & Weaver, K. F. (2016a). Col-
onizing the Caribbean: biogeography and evolution of livebearing
fishes of the genus Limia (Poeciliidae). Journal of Biogeography,43,
1808–1819.
Weaver, P. F., Tello, O., Krieger, J., Marmolejo, A., Weaver, K. F.,
Garcia, J. V., & Cruz, A. (2016b). Hypersalinity drives physiological and
morphological changes in Limia perugiae (Poeciliidae). Biology Open,5,
1093–1101.
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Rodriguez-Silva R, Weaver PF. A new
livebearing fish of the genus Limia (Cyprinodontiformes:
Poeciliidae) from Lake Miragoane, Haiti. J Fish Biol. 2020;96:
1360–1369. https://doi.org/10.1111/jfb.14301
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