TEXAS TECH UNIVERSITY

Natural Science Research Laboratory

Occasional Papers

Museum of Texas Tech University

Number 339 2 August 2016

Patterns of Genetic Diversification in a Widely Distributed Species or

Bat, Molossus molossus

Laramie L. Lindsey and Loren K. Ammerman

Abstract

The taxonomy and evolutionary relationships of the Velvety Free-tailed Bat, Molossus
molossus , from Central and South America long have been debated. Within this species, and
in fact the entire genus Molossus , specimens have been difficult to identify and have presented
several taxonomic challenges. The objective of this project was to characterize the genetic
relationship among individuals representing subspecies of the widely distributed species, M.
molossus. We tested the hypothesis that genetic patterns of diversification would reflect subspe¬
cies lineages. The mitochondrial gene cytochrome b (cyt b) was amplified and sequenced for
specimens throughout its geographic range. A Bayesian analysis of 678 base pairs of the cytZ>
gene was conducted for 65 specimens with M. aharezi as an outgroup. Our results showed
that the subspecies M. m. daulensis, recognized based on morphology and geographic location,
formed a statistically supported mitochondrial lineage in the phylogenetic analysis. However,
not all currently recognized subspecies of M. molossus were recovered by this analysis. One
lineage, M. m. tropidorhynchus from Cuba, formed a divergent monophyletic lineage. Overall,
the average divergence across all specimens was 4.7%; however the M. m. tropidorhynchus
lineage was 7.9% divergent from the other M. molossus specimens. This level of divergence
and the recovery of a monophyletic lineage containing all Cuban specimens was consistent with
recognition of the taxon as a distinct species.

Key words: cytochrome b, genetic species, Molossus molossus , systematics, taxonomy

Introduction

The Velvety Free-tailed Bat is a widely distrib¬
uted species in the family Molossidae. They reside
in tropical and temperate areas of Central and South
America and the Greater and Lesser Antilles Islands
(Simmons 2005). Since the first molossid was de¬
scribed as Vespertilio molossus by Pallas (1766), large

numbers of species and subspecies have been assigned
to the genus Molossus (Table 1). Sexual dimorphism
and high degrees of local variation in Molossus have
confounded species definitions and groupings (Dolan
1982), and therefore the taxonomy and phylogenetic re¬
lationships of Molossus lineages remain highly debated.

2

Occasional Papers, Museum of Texas Tech University

Table 1. Summary of taxonomic history of Molossus species. Species names marked with the same symbol highlight
synonymous groupings of the currently recognized Molossus species. An additional species, M. alvarezi , was described
by Gonzalez-Ruiz et al. (2011) from Yucatan, Mexico.

Miller (1913)

Dolan (1989)

Simmons (2005)

Eger (2007)

M. rufus°

M. rufus°

M. rufus°

M. rufus°

M. nigricans 0

M. pretiosus *

M. pretiosus*

M. pretiosus*

M. pretiosus*

M. sinaloae A

M. sinaloae A

M. sinaloae A

M. sinaloae A

M. currentium

M. currentium

M. currentium

M. bondae'

M. bondae'

M. bondae'

M. aztecus*

M. aztecus*

M. aztecus*

M. barnesi #

M. barnesi*

M. coibensis*

M. coibensis*

M. coibensis*

M. coibensis*

M. major (M molossusf

M. fulginosis %

M. verrilli %

M. fortis %

M. debilis %

M. obscurus %

M. crassicaudatus %

M. pygmaeus %

M. tropidorhynchus %

M. molossus %

M. molossus %

M. molossus %

Miller (1913) recognized 18 species in the genus
Molossus by morphologically comparing Molossus
specimens residing in the United States National Mu¬
seum. He did not recognize M. molossus but instead
split this widespread species into nine species. Dolan
(1989), in the most recent treatment of the diversity
within Molossus , recognized seven of Miller’s origi¬
nal 18 species. Dolan combined nine species into the
single taxon M. molossus (Table 1). The type specimen
named by Pallas (1766) remains the type specimen for
the species M. molossus although it is a lectotype based
on Husson (1962), who examined Vespertilio molos¬
sus. More recently, Simmons (2005) recognized eight
species in the genus, whereas Eger (2007) recognized
seven species. Eger (2007) placed both M. barnesi
and M. aztecus in M. coibensis. Also, Eger (2007)

recognized M. bondae as a separate species from M.
currentium based on pelage differences. A recent mor¬
phometric study (Gonzalez-Ruiz et al. 2011) discovered
a new species from the Yucatan Peninsula in Mexico;
M. alvarezi is similar to M. sinaloae, but some cranial
measurements do not overlap and the two species are
geographically separated (Gonzalez-Ruiz et al. 2011).

Based on morphological data, Dolan (1989)
concluded that species of Molossus are differentiated
primarily on the basis of size; however, many popula¬
tions within a single species also differed in size based
on their geographic locality. According to Dolan
(1989), “all species of Molossus exist in numerous,
morphologically discrete populations and can be con¬
sidered polytypic”; this suggests morphological data

Lindsey and Ammerman—Genetic Diversification of Molossus molossus

3

alone is unreliable for differentiating lineages. Further,
Dolan (1989) and Warner et al. (1974) were not able
to uncover any interspecific variation among M. rufus ,
M. molossus , and M. sinaloae using a chromosomal
approach; all three species had the same karyotype.
In addition, Dolan (1989) constructed a phenogram
based on electrophoretic data of 11 polymorphic al-
lozyme loci, and these data were unable to resolve
the relationship of M. rufus and M. pretiosus. Only
M. bondae had a single species-specific marker allele.
Therefore, Dolan referred to the M. rufus , M. pretiosus ,
and M. bondae clade as the rufus complex. Molossus
molossus possessed two species-specific markers and
was sister to the rufus complex. Molossus sinaloae
was the most divergent taxon in Dolan’s analysis, as
it was basal to all other taxa. Dolan’s results did not
support a strong correlation between electrophoretic
data and geographic proximity, suggesting isolated
populations and possible subspecies in many of the
species of Molossus that she examined.

Molossus molossus and its subspecies remain
the most highly debated group in the genus, having
about 20 synonyms (Simmons 2005). The diversity of
recognized forms could be a result of the widespread
geographic distribution of this taxon compared to other
Molossus species. Complex patterns of intraspecific
variation across the geographic range make resolving
M. molossus taxonomy difficult. Eger (2007) suggested

that a complete review of the entire M. molossus group
was needed to clarify the status of the numerous avail¬
able names. Genoways et al. (1981) also suggested
genetic analyses would be informative and beneficial
for M. molossus.

Nine of the original species recognized by Miller
(1913) have been placed within M. molossus and many
of these are now recognized as subspecies (Table 2).
Simmons (2005) and Eger (2007) differed on recogni¬
tion of subspecies of M. molossus. Simmons (2005)
recognized seven subspecies: M. m. molossus , M. m.
debilis , M. m. pygmaeus, M. m. fortis , M. m. milleri ,
M. m. tropidorhynchus, and M. m. verrilli. The four
subspecies recognized by Eger (2007) were M. m.
molossus , M. m. pygmaeus , M. m. daulensis, and M.
m. crassicaudatus. Simmons (2005) and Eger (2007)
only agreed on two subspecies, M. m. molossus and M.
m. pygmaeus. Both Simmons (2005) and Eger (2007)
placed all of the unresolved subspecies within M. m.
molossus.

Genoways et al. (1981) attempted to decipher
intra-island and inter-island variation of M. molossus
from three Antillean Island populations using morpho¬
logical data. Specimens from Jamaica, Guadeloupe,
and Trinidad were examined to determine local versus
geographic variation. From all specimens, one external
and nine cranial measurements were recorded (Ge-

Table 2. Subspecies of Molossus molossus recognized by Simmons (2005) and/or Eger (2007) and type localities as¬
sociated with each subspecies.

Subspecies

Authority

Type Locality

M. m. crassicaudatus

Geoffroy 1805

Asuncion, Central Paraguay

M. m. daulensis

Allen 1916

“Daule”, Los Rios, Ecuador

M. m. debilis

Miller 1913

St. Kitts, Lesser Antilles

M. m. fortis

Miller 1913

“Luquillo”, Puerto Rico

M. m. milleri

Johnson 1952

Bermuda

M. m. molossus

Pallas 1766

Martinque, West Indies

M. m. pygmaeus

Miller 1900

Netherlands Antilles

M. m. tropidorhynchus

Gray 1839

Cuba

M. m. verrilli

Allen 1908

“Samana”, Dominican Republic

4

Occasional Papers, Museum of Texas Tech University

noways et al. 1981). They concluded that there was
significant morphological variation among intra-island
populations, as well as inter-island variation for popula¬
tions of M. molossus. Genoways et al. (1981) suggested
that a high degree of philopatry and inbreeding was the
reason for the high levels of local geographic variation
seen in this species.

Examining genetic patterns of specimens of M.
molossus from various geographic localities should
help clarify unresolved relationships. Few studies have
been conducted to determine relationships among sub¬
species of M. molossus , especially any using a genetic
approach. McDonough et al. (2011) used the mito¬
chondrial gene cytochrome oxidase I (COI) to identify
Molossus specimens but did not address subspecies
relationships. Furthermore, Sudman et al. (1994) used a
cytb sequence from M. molossus to identify the familial
affinity of Tomopeas ravus ; however, there have been
no further genetic analyses using DNA sequence data

to determine patterns of genetic divergence within M.
molossus. More recently, Gager et al. (2016) used 18
microsatellite markers and the mitochondrial gene
COI, along with morphological and acoustic data, to
distinguish between morphologically similar species of
M. bondae , M. molossus , and M. coibensis in Panama
(Gager et al. 2016). M. bondae was identified based
on size and pelage differences, while M. molossus and
M. coibensis were differentiated using the microsatel¬
lite markers and COI (Gager et al. 2016). The authors
suggest using multiple approaches to determine species
that are morphologically similar. The lack of published
genetic data for species of Molossus is apparent, and
further studies of the phylogenetic relationships of
this genus are needed. The objective of this study was
to use the mitochondrial gene cytochrome b (cytZ>) to
characterize patterns of diversification within M. molos¬
sus. We tested the hypothesis that genetic patterns of
diversification would reflect subspecies lineages.

Methods and Materials

Molecular methods. —Tissues of M. molossus
were borrowed from the Angelo State Natural History
Collection, the Field Museum of Natural History in
Chicago, and the Natural Science Research Fabora-
tory, Museum of Texas Tech University. Specimens
were selected to cover the species range from Central
America into northern and central South America and
a few sites within the Fesser and Greater Antillean
Islands (Fig. 1). A morphological key provided by
Eger (2007) was used to confirm species identifica¬
tions of selected specimens included in the genetic
analysis. DNA was extracted from heart, kidney, liver,
or muscle tissues that were either frozen or in lysis
buffer using the DNeasy Tissue Kit (QIAGEN Inc.,
Valencia, California) following manufacturer’s proto¬
col. The mitochondrial cytb gene was amplified using
the polymerase chain reaction (PCR); 12.5pF reactions
contained 30-60 ng of DNA, 1 unit of Taq polymerase
(New England BioFabs, Ipswich, MA), 2.5 mM of each
dinucleoside triphosphate, IX Taq buffer, 1.5-2.0 mM
MgCl 2 and 0.16 pM of forward and reverse primers.
Cytb was amplified with the following thermal profile;
1 cycle at 94°C for 2 min; 39 cycles at 94°C, 48°C,
and 72°C for 1 min each; and a final extension cycle

at 72°C for 10 min. PCR and sequencing of each gene
fragment was carried out using combinations of primer
15547 (5’- GGCAAATAGGAAATATCATTC-3’;
Edwards et al. 1991), primer Gludg (5’-TGACTT-
GAARAACCATCGTTG-3’; Palumbi 1996), primer
MVZ04 (5 ’-GCAGCCCCTCAGAATGATATTTGT-3 ’;
Smith and Patton 1991), and primer MVZ05 (5’-
C G A AGC TT G ATAT G A A A A AC CAT C GT T G- 3 ’;
Smith and Patton 1991). PCR products were sequenced
using GenomeFab DTCS-Quick Start Mix in a Beck¬
man Coulter CEQ8000 automated sequencer following
manufacturer’s protocol, except a quarter of the recom¬
mended volumes were used.

Phylogenetic analyses. —Sequencher version 5.0
(Gene Codes Corporation, Ann Arbor, Michigan) and
MEGA5 (Tamura et al. 2011) were used to align the
sequences, which were then refined by eye if needed.
Furthermore, we confirmed that all cytb fragments
translated to amino acid sequences. Sequences were
deposited in GenBank (Appendix). Models with the
lowest Bayesian Information Criterion were used to
describe the substitution pattern that best fit the data
set (Tamura et al. 2011). AMaximum Fikelihood (ME)

Lindsey and Ammerman—Genetic Diversification of Molossus molossus

5

Figure 1. Map of Central and South America and Caribbean Islands with locations of specimens of Molossus
obtained for this study. Shapes correspond to species and subspecies designation (diamond = M. rufus , square = M. m.
daulensis, circle = M. m. crassicaudatus , and triangle = M. m. molossus (includes M. m. debilis , M. m. fortis , and M.
m. tropidorhynchus according to the taxonomy recognized by Eger 2007)).

6

Occasional Papers, Museum of Texas Tech University

tree was generated in MEGA5. Statistical nodal support
was evaluated with 1000 bootstrap pseudoreplicates.

A Bayesian analysis (BI) of cytb was performed
using MrBayes version 3.1.2 (Ronquist and Huelsen-
beck 2003). Analyses consisted of two simultaneous
runs, each with four Markov Chain Monte Carlo chains
(three heated and one cold) run for five million genera¬
tions. Convergence of the two runs was determined
when convergence diagnostic <0.01. Trees were
sampled every 100 generations with a 25% burn-in. A
50% majority rule consensus tree was used to calculate
posterior probabilities and included the proportion of
trees saved after convergence of likelihood scores was
reached. Nodes in resulting trees containing >0.95
Bayesian posterior probabilities (BPP) were considered
statistically significant (Ronquist and Huelsenbeck,
2003). FigTree v. 1.4.0 (http://tree.bio.ed.ac.uk/soft-
ware/figtree/) was used to visualize and draw trees
generated by MrBayes.

Patterns of diversification recovered by the
phylogenetic analysis were interpreted based on two
different criteria. Genetic divergences between clades
were evaluated for agreement with divergence levels
outlined by Baker and Bradley (2006) for sister species
of mammals by using the Kimura 2-parameter model
without gamma correction. These divergences be¬
tween clades were calculated in MEGA5. In addition,
sister clades resulting from ML and BI analyses were
compared using the K/0 method introduced by Birky
(2013) to delimit lineages that should be recognized as
species. The steps outlined in Birky (2013) were used
to calculate the value of K/0, which is the ratio of the
mean pairwise sequence difference between a pair of
clades (K) and the mean pairwise difference within a
clade (0). A pair of clades was considered to be differ¬
ent species if K/0 > 4.

Results

A total of 63 novel cytb sequences (Appendix)
of Molossus species from various geographic locations
were included in the phylogenetic analysis (Fig. 2).
The final alignment included 678 base pairs of original
sequences from 4 M. rufus , 2 M. aharezi , 1 M. bondae,
2 M. coibensis, and 54 M. molossus (Appendix). Two
M. molossus sequences from GenBank also were in¬
cluded in the alignment (JQ915205.1 and L19724.1).
Both the Bayesian and ML methods recovered similar
topologies (Fig. 2). The ML tree was generated us¬
ing the model Tamura 3-parameter with gamma rate
distribution (alpha = 0.1659, Log likelihood score =
-1155.248). The species M. molossus did not form a
monophyletic lineage. Four specimens ofM molossus
from Cuba (Fig. 2, Clade D) clearly formed a separate
lineage from the rest of the M. molossus specimens
(Clade Al-3) with high BPP support (1.0). The ma¬
jority of M. molossus specimens (Clade A2) clustered
together in a large polytomy with the exception of two
distinct lineages that were strongly supported—one
from the western slope of the Andes in Ecuador and
northern Peru (Clade Al; BPP 1.0) and the other from
Cuba (Clade D; BPP 1.0). There was not significant
support for resolution of branching order among clades
Al, A2, A3, and B+C. Subspecies M. m. fortis (plus

M. m. debilis ) from Puerto Rico and St. Kitts/St. Croix,
M. m. daulensis from western Ecuador and Peru, and
M. m. tropidorhynchus from Cuba formed statistically
supported monophyletic lineages in this phylogenetic
analysis (Fig. 2). All other currently recognized sub¬
species (Table 2), including M. m. crassicaudatus from
South America, did not form separate lineages.

The subspecies M. m. daulensis (Clade A1), from
the western slope of the Andes in Ecuador and Peru,
had an average genetic divergence of 2.7% from the
eastern Ecuadorian specimens and an average genetic
divergence of 3.7% from M. m. molossus specimens
(Clade A2; Table 3). The clade containing the Brazil/El
Salvador specimens (Clade A3) had an average genetic
divergence of 6.0% from the rest of the M. molossus
clade (Clade A1/A2; Table 3). Furthermore, a lineage
containing the currently recognized Cuban subspecies
M. m. tropidorhynchus (Clade D) had an average ge¬
netic divergence of 7.9% from the other M. molossus
(Clade Al, A2, A3) included in the study (Table 3).

The group containing M. rufus!M. bondae (Clade
B) had an average genetic divergence of 8.4% from
M. molossus (all members of Clade Al, A2, A3). The

Lindsey and Ammerman—Genetic Diversification of Molossus molossus

1

L Tl.

TK32043 M. m. tropidorhynchns CU
TK32141 M. m. tropidorhynchns CU
■TK32142M m. tropidorhynchus CU
TK32081 M. m. tropidorhynchus C U
TK19170 M. coibensis VZ

if-

TK19168 M coibensis VZ

- TK9218 M. bondae JM
-TK56709 M. rufus PY

- TK86608 M rufus GY

- TK86649 M. rufus GY
■ TK86648 M. rufus GY

£

BDP3265 M. m. molossus BR
BDP3280 M. m. molossus BR
BDP3271 M. m. molossus BR

TK34864 M. m. molossus SV
TK34865 M. m. molossus SV

-TK17231 M. m. molossus SR

-TK17232 M. m. molossus SR

■ TK86604 M. m. molossus GY
— TK86607 M. m. molossus GY

— TK86651 Mm. molossus GY

— TK86653 M. m. molossus GY

■ TK86655 M. m. molossus GY

— TK64488 M. m. molossus PY
— ASK7730 M. m. molossus EC

42

0 . 9 /- 1— i— FMNH213845 M. m. molossus EC
FMNH213847 M. m. molossus EC
-776 - TK14589 M. m. molossus BO
- TK86602 M. m. molossus GY
r ASK7759 M. m. molossus EC
TK11344 M. m. molossus SR

■ ASK7760 M. m. molossus GY
r- FMNH206530 M m.fords PR
■ FMNH206531 M. m.fords PR

0 . 98 /- —■ FMNH206532 M. m. fords PR
|- J0915205.1 M. m. debilis KN
TK148718 M. m. molossus VI
TK148739 M. m. molossus VI
- FMNH213850 M. m. molossus EC
TK11333 M. m. molossus SR

- TK18548 M. m. molossus GD
— TK18565 M. m. molossus GD

TK18622 M m. molossus VC

■ TK18651 M. m. molossus VC
— TK64799 M m. molossus PY

TK86603 M. m. molossus GY
TK86605 M m. molossus GY
TK86625 M. m. molossus GY
TK151406 M m. molossus BB
TK151439 M. m. molossus BB
— TK86654 M. m. molossus GY

- TK128560 M. m. molossus VC
TK128561 M m. molossus VC

- TK151281 M. m. molossus LC
TK161199 M. m. molossus LC

— TK161331 M m. molossus LC

LI9724.1 M. m. daulensis PE

1 . 00/85

- ASK7787 M m. daulensis EC

- ASK7779 M. m. daulensis EC

-L

H - ASK7786 M. m. daulensis EC
TK134642 M m. daulensis EC
_r TK134887 M m. daulensis EC
TK135112 M m. daulensis EC
FN32915 M. alvarezi MX
FN32916 M alvarezi MX

Clade D
I Clade C
Clade B

I Clade A3

Clade A2

CladeA1

0.3 substitutions/site

Figure 2. Bayesian phylogram of M. molossus specimens based on 678 base pairs of the cyt b gene,
rooted with the outgroup M. alvarezi. Nodes are labeled with BPP values followed by bootstrap
values (— if BPP values <0.95 or if bootstrap values <70). Clades Al, A2, and A3 represent the
species M. molossus. BB=Barbados, BO=Bolivia, BR=Brazil, CU=Cuba, EC=Ecuador, GD=Grenada,
GY=Guyana, JM=Jamaica, KN=St. Kitts and Nevis, LC=St. Lucia, MX=Mexico, PE=Peru, PR=Puerto
Rico, PY=Paraguay, SR=Suriname, SV=E1 Salvador, VC=St. Vincent and the Grenadines, VI=US
Virgin Islands, St. Croix, VZ=Venezuela.

8

Occasional Papers, Museum of Texas Tech University

Table 3. Average Kimura 2-parameter distances between and within subspecies of M. molossus (Clade Al, A2, A3,
D) and within and between species M. rufus/M. bondae (Clade B), M. coibensis (Clade C), and outgroup M. alvarezi
based on 678 bases of cyt b. Sample sizes are listed in parentheses. Clade labels also are identified in Fig. 2.

Al

A2

A3

B

C

D

Outgroup

Clade Al (7)

0.0062

Clade A2 (40)

0.0373

0.0111

Clade A3 (5)

0.0672

0.0532

0.0130

Clade B (5)

0.0942

0.0805

0.0781

0.0246

Clade C (2)

0.0880

0.0554

0.0610

0.0587

0.0047

Clade D (4)

0.0816

0.0752

0.0809

0.1352

0.1097

0.0026

Outgroup (2)

0.1487

0.1370

0.1394

0.1164

0.1326

0.1270

0.0030

Venezuelan specimens (M coibensis, Clade C) had an
average genetic divergence of 6.8% from Clade Al,
A2, and A3 combined and a genetic divergence of 5.9%
from Clade B. The outgroup, M. alvarezi, had an aver¬
age genetic divergence of 13.4% from the rest of the
specimens included in this study (Table 3).

K/0 values were generated for six clade pairings
using the methods described by Birky (2013) to assess
species limits (Table 4). Based on criteria outlined by
Birky (2013), K/0 ratios greater than 4 were consid¬

ered different species. In our analysis, this measure
supports species status for the Cuban clade (Clade D)
separate from M. molossus (Clades designated by A),
M. rufus/M. bondae (Clade B) separate from the Ven¬
ezuelan clade of M. coibensis (Clade C), and M. rufus
(Clade B) separate from M. molossus clade (Clades
designated by A). However, the Brazil/El Salvador
clade (Clade A3) does not have a K/0 ratio greater
than 4, indicating that the Brazilian and Salvadoran
samples should be considered part of M. molossus
based on cyt b data.

Table 4. K/0 ratios used to determine species delimitation for two different clades. K/0 > 4 are considered separate
species while K/0 < 4 are considered the same species (Birky 2013). Clade labels are identified in Fig. 2.

Clade 1

Clade 2

K/0

Clade B (M rufus/M. bondae)

Clade C (M. coibensis)

4.2*

Clade A1 (M. m. daulensis)

Clade A2 (M m. molossus)

2.2

Clade D (M m. tropidorhynchus)

Clade A1/A2/A3 (M molossus)

4.1*

Brazil

El Salvador

3.3

Clade A3 (Brazil/El Salvador)

Clade A1/A2 (M molossus)

2.8

Clade B (M rufus/M. bondae)

Clade A1/A2/A3 (M molossus)

4.3*

Lindsey and Ammerman—Genetic Diversification of Molossus molossus

9

Discussion

Phylogenetic patterns recovered from the cyt b
analysis of M. molossus specimens were not consistent
with all currently recognized subspecies designations.
However, three lineages were consistent with currently
recognized subspecies of M. molossus , including M. m.
daulensis from west of the Andes in Ecuador and Peru,
M. m. tropidorhynchus from Cuba, and M. m. fortis ,
the subspecies from Puerto Rico, with St. Croix and
St. Kitts samples (M m. debilis). Molossus m. fortis
and M. m. debilis were recognized originally by Miller
(1913) based on morphology and geographic location.
Our genetic results suggest that these subspecies,
limited to Puerto Rico and the Virgin Islands (Gannon
et al. 2005), should be synonymized. Despite a few
well-supported clades, there is very little significant
phylogenetic structure to resolve branching order of
the five species of Molossus examined in this study.
These results suggest that there is some consistency
between these genetic data and the currently recognized
subspecies, but the cytb marker was largely unable
to recover a monophyletic M. molossus clade due to
low divergence between this species and M. rufus , M.
bondae , and M. coibensis.

M. m. tropidorhynchus should not be considered
a subspecies of M. molossus based on our results.
Instead, the Cuban specimens (Clade D) should be
elevated to species level, M. tropidorhynchus. In 1839,
Gray described a holotype, probably from Havana,
Cuba, as M. tropidorhynchus (Carter and Dolan 1979).
M. tropidorhynchus is reported to be somewhat smaller
than M. molossus from Central and South America
and to have an olive brown pelage (Silva-Taboada
1979). Frank (1997) described the occurrence of M.
m. tropidorhynchus from the Florida Keys. Thus, the
specimens of M. molossus in the United States are
most likely M. tropidorhynchus and not M. molos¬
sus , although this remains to be tested. No further
information on the distribution of M. tropidorhynchus
is known. We compared forearm measurements and
the second phalanx on the fourth digit of two Cuban
specimens to published keys to determine they were
M. tropidorhynchus and not another molossid species
(Miller 1904; Silva-Taboada 1979).

According to Bradley and Baker (2001), cytb
genetic divergence values within the 2-11% range have

a high probability of representing separate species of
mammals. Applying the Genetic Species Concept,
first proposed by Bateson (1909) and later redefined
by Baker and Bradley (2006), M. tropidorhynchus
exhibits reciprocal monophyly and a cytb divergence
value of 7.9% from all other M. molossus specimens,
consistent with recognition of the taxon as a species
(Baker and Bradley 2006). Additional justification
for elevating this species comes from the criteria used
by Birky (2013); the Cuban clade (Clade D) had a
K/0 ratio greater than 4 (Table 4). Although the K/0
technique primarily has been used to determine specific
relationships in asexual organisms, Birky proposed that
use for determining species limits in vertebrates is also
possible. Baker and Bradley (2006) suggested that, if
possible, it is important to have both nuclear and mito¬
chondrial markers to document presence or absence of
species. Haplotypes of cyt&, like all other mitochon¬
drial genes, represent lineages of a maternally inherited
marker and should be used cautiously to represent spe¬
cies relationships. Furthermore, Davalos and Russell
(2014) caution that sex-biased dispersal could mislead
interpretations of mitochondrial patterns. However,
“barcoding genes” such as COI also are mitochondrial
genes and are widely used for species identification
(Hajibabaei et al. 2007; Clare et al. 2011), validat¬
ing our approach. Furthermore, mitochondrial genes
have been used in several other mammalian species to
determine very closely related species and subspecies
designations. Many studies have used cytb and/or other
mitochondrial genes to examine very closely related
species of Peromyscus (Harris et al. 2000; Bradley et
al. 2007) and have reported similar genetic divergences
among closely related rodent taxa. Piaggio et al. (2002)
examined two mitochondrial markers to determine that
Myotis occultus is not a subspecies of Myotis lucifugus
as previously reported. Sun et al. (2008) combined cytb
sequences, morphological and phonic data to determine
subspecies relationships of Rhinolophus macrotis in
China. More recently, Sun et al. (2015) used the whole
mitochondrial genome to examine the relationship of
two subspecies of Rhinolophus sinicus. Hence, adding
nuclear data from a rapidly evolving marker should as¬
sist in confirming our proposal that M. tropidorhynchus
be recognized as a separate species from M. molossus.

10

Occasional Papers, Museum of Texas Tech University

Clade B and C containing M. rufus , M. coibensis,
and M. bondae specimens unexpectedly produced a
paraphyletic M. molossus when the Cuban specimens
are considered as a subspecies of M. molossus. The
position of Clade B/C was unresolved and there¬
fore questions remain regarding the relationships of
other Molossus species. Unfortunately, according to
personnel at the USNM, the voucher specimens for
USNM582416/TK86608, USNM582418/TK86648,
and USNM582419/TK86649 have been misplaced
so we were not able to examine or positively identify
these three specimens. Based on their tight clustering
with one known representative of M. rufus (TTU96091/
TK56709), and the fact that M. rufus is known to oc¬
cur in Guyana (Eger 2007), we suspect that they are
M. rufus. The only Jamaican specimen (CM44668/
TK9218) included in this study is placed in the same
clade as M. rufus (Clade B) in the cytb tree. However,
according to Genoways et al. (2005), the only species
of Molossus to occur on the island of Jamaica is M.
molossus. We re-examined this specimen and, based
on published keys (Burnett et al. 2001; Eger 2007),
the specimen is M. bondae. Dolan (1989) placed M.
bondae as sister to M. rufus\ our tree depicts a similar
relationship. Therefore, CM44668/TK9218 represents
M. bondae ; and, to our knowledge is the first record of
M. bondae from Jamaica. However, given the uncer¬
tainties uncovered regarding the identity of several of
our specimens, a comprehensive phylogenetic analysis
of the entire genus is critically needed.

The occurrence of individuals from Brazil and El
Salvador in a single, although unsupported, clade (A3)
was unexpected, given that they are geographically well
separated. We do not know whether to consider this
clade as part of M. molossus. Clade A3 (Brazil + El
Salvador) had a genetic divergence of 6.0% from other
M. molossus specimens, which could be interpreted as
a species level divergence (Bradley and Baker 2001).
However, K/0 values do support El Salvador and Brazil
(Clade A3) as the same species as the rest of M. molos¬
sus (Clade Al and A2). Another possibility is that
the Brazilian specimens in Clade A3 could represent
M. currentium. This species is known from northern
Paraguay and is only slightly larger than M. molossus
(Eger 2007), making morphological identification
challenging. Detailed morphological work on these
specimens was not possible and therefore research
on additional specimens from Brazil and El Salvador

will be necessary to clarify the confusion regarding the
identity of the specimens in Clade A3.

Low genetic divergence values (1.2%) were
recorded for M. molossus specimens (Clade A2) over
a wide geographic area. The low genetic divergence
within M. molossus suggests that this group of bats
evolved relatively recently. Larsen et al. (2007)
observed a similar lack of geographic structuring in
the Caribbean and South American species, Artibeus
planirostris , and hypothesized rapid radiation and dis¬
persal for this species. Rapid radiation and dispersal
could account for the lack of geographic structuring
within M. molossus as well. Molossus molossus have
been reported to have excellent colonizing ability and
a capacity for overwater dispersal (Frank 1997), as
demonstrated by their colonization of Florida in recent
history. Other molossids are known to forage over
long distances (up to 50 mi (80 km) in Eumopsperotis,
Vaughan 1978) or to make long distance migrations
(Tadarida brasiliensis. Glass 1958; Cockrum 1969;
Russell et al. 2005), so dispersal to Caribbean islands
would presumably not be difficult for M. molossus.

Molossus molossus is a difficult species to iden¬
tify because of high morphological variation across
the species range (Genoways et al. 1981; Dolan 1989).
Small localized demes and environmental constraints
could have played a role in increasing morphological
variation. However, despite the fact that this variation
is reflected in numerous recognized subspecies, geneti¬
cally these bats are quite uniform (with the exception of
the M. tropidorhynchus lineage). Phenotypic plasticity
could also explain the high degree of morphological
variation co-occurring with apparent genetic uniformity
in M. molossus. Phenotypic plasticity is described as
the capacity of a single genotype to exhibit variable
phenotypes in different environments (Whitman and
Agrawal 2009). As M. molossus populations expanded,
the species may have adapted to different environmen¬
tal factors, resulting in morphological variation that is
not reflected in the mitochondrial marker examined in
this study.

Biogeographically, the results of this study sup¬
port two invasions into the Caribbean, as well as a
separation of populations by the rise of the Andes. We
hypothesize an older dispersal event into Cuba by the
ancestor of M. tropidorhynchus and a younger dispersal

Lindsey and Ammerman—Genetic Diversification of Molossus molossus

11

event into the Lesser Antillean Islands by M. molos¬
sus. Cuba, Hispaniola, and Jamaica are much older
islands that originated from the Caribbean plate when
South America and North America started to separate
from each other (Davalos 2009). Furthermore, smaller
islands were submerged during periods of high sea
levels while Cuba and Hispaniola were a single land
mass (Davalos 2009). This may explain why Cuba
has many endemic species, including the bat species
Lasiurus insularis , Mormopterus minutus, Natalus
primus (Griffiths and Klingener 1988; Davalos 2004)
and now Molossus tropidorhynchus.

M. m. daulensis from the western slope of the
Andes Mountains appears to have been separated from
M. m. molossus on the eastern slope for a sufficient
length of time to accumulate distinct genetic differences
(3.7% divergence at cytb). These results are similar to
the divergence seen in COI sequences of M. molossus
bats from east and west of the Andes by McDonough
et al. (2011). Furthermore, other species of bats such
as Eumops wilsoni and E. glaucinus show similar al-
lopatric distribution and level of divergence based on

their location on either side of the Andes (Bartlett et al.
2013). At this time, we are not proposing the elevation
of M. m. daulensis to species status; however future
work, such as a population genetic approach, might be
appropriate for examining this lineage more closely.

The phylogeny of M. molossus generated in this
study is intended to serve as a working hypothesis for
future work on the hidden biodiversity in this species.
Further investigation should be carried out on the re¬
maining three subspecies in the species M. molossus
that we were unable to include in this study. Likewise,
future studies should include representatives from all
recognized species of Molossus to create a clearer pic¬
ture of the evolutionary relationships in this problematic
genus. Sequence data from additional specimens and
from a more rapidly evolving genetic marker, such as
microsatellites (Gager et al. 2016), or high-throughput
sequencing analyses, such as RADSeq (Davey and
Blaxter 2010), should give a more accurate representa¬
tion of the diversity within M. molossus across Central
and South America and the Caribbean.

Acknowledgments

Many thanks to the Angelo State Natural History
Collection, the Museum of Texas Tech University,
and the Field Museum of Natural History for loaning
specimen tissues. Thank you to Heath Gamer, Suzanne
McLaren, and Darrin Lunde for tracking down voucher
specimens and finding measurements to help provide
morphological identifications for select specimens.
Special thanks to Hugh H. Genoways, Gary G. Kwie-
cinski, Roxanne J. Larsen, Peter A. Larsen, Tom Lee,
Jessica Light, Bruce D. Patterson, Scott C. Pedersen,

and Vicky J. Swier for collecting and loaning tis¬
sues for this study. Thank you to Robert D. Bradley,
Christopher Dunn, Megan C. Keith, Celia Lopez-
Gonzalez, Roxanne J. Larsen, Peter A. Larsen, Molly
M. McDonough, Nicte Ordonez-Garza, and Emma K.
Roberts for providing input and advice for this project.
We would like to thank the Center for Innovation in
Teaching and Research at Angelo State University for
granting the Graduate Research Fellowship in support
of this research.

Literature Cited

Baker, R. J., and R. D. Bradley. 2006. Speciation in mam¬
mals and the genetic species concept. Journal of
Mammalogy 87:643-662.

Bartlett, S. N., M. M. McDonough, and L. K. Ammerman.
2013. Molecular systematics of bonneted bats
(Molossidae: Eumops ) based on mitochondrial and
nuclear DNA sequences. Journal of Mammalogy
94:867-880.

Bateson, W. 1909. Heredity and variation in modern lights.
Pp. 85-101 in Darwin and Modern Science (A. C.
Seward, ed.). Cambridge University Press, Cam¬
bridge, United Kingdom.

Birky, C. W., Jr. 2013. Species detection and identification
in sexual organisms using population genetic
theory and DNA sequences. PLoS ONE 8:e52544.
doi:10.1371/journal.pone.0052544.

12

Occasional Papers, Museum of Texas Tech University

Bradley, R. D., and R. J. Baker. 2001. A test of the Genetic
Species Concept: Cytochrome-Z) sequences and
mammals. Journal of Mammalogy 82:960-973.

Bradley, R. D., N. D. Durish, D. S. Rogers, J. R. Miller, M.
D. Engstrom, and C. W. Kilpatrick. 2007. Toward
a molecular phylogeny for Peromyscus : Evidence
from mitochondrial cytochrome-Z) sequences.
Journal of Mammalogy 88:1146-1159.

Burnett, S. E., J. B. Jennings, J. C. Rainey, and T. L. Best.
2001. Molossus bondae. Mammalian Species
668:1-5.

Carter, D. C., and R G. Dolan. 1979. Distributional notes and
records of Middle American Chiroptera. Journal of
Mammalogy 60:644-649.

Clare, E. L., B. K. Lim, M. B. Fenton, and P. D. N. Hebert.
2011. Neotropical bats: Estimating species diver¬
sity with DNA barcodes. PLoS ONE 6:e22648.
doi: 10.13 71 /j ournal. pone.0022648.

Cockrum, E. L. 1969. Migration in the guano bat, Tadarida
brasiliensis. Miscellaneous Publications, The
University of Kansas Museum of Natural History
51:303-336.

Davalos, L. M. 2004. Phylogeny and biogeography of Carib¬
bean mammals. Biological Journal of the Linnean
Society 8:373-394.

Davalos, L. M. 2009. Earth history and the evolution of Ca¬
ribbean bats. Pp 96-115 in Island Bats: Evolution,
Ecology and Conservation (T. H. Fleming and P.
A. Racey, eds). University Chicago Press. 592 pp.

Davalos, L. M., and A. L. Russell. 2014. Sex-biased dispersal
produces high error rates in mitochondrial distance-
based and tree-based species delimitation. Journal
of Mammalogy 95:781-791.

Davey, J. W., and M. L. Blaxter. 2010. RADSeq: Next-gen¬
eration population genetics. Briefings in Functional
Genomics 9:416-423.

Dolan, P. G. 1982. Systematics of Middle American mastiff
bats {Molossus). Doctoral Dissertation, Texas Tech
University, Lubbock.

Dolan, P. G. 1989. Systematics of Middle American mastiff
bats of the genus Molossus. Special Publications,
Museum of Texas Tech University 29:1-71.

Edwards, S. V., P. Arctander, and A. C. Wilson. 1991.
Mitochondrial resolution of a deep branch in the
genealogical tree for perching birds. Proceedings of
the Royal Society of London 243:99-107.

Eger, J. L. 2007. Family Molossidae P. Gervais, 1856. Pp
399-439 in Mammals of South America, Volume

1: Marsupials, Xenarthrans, Shrews, and Bats. 4 th
ed. (A. L. Gardner, ed.). University of Chicago
Press. 689 pp.

Frank, P. A. 1997. First record of Molossus molossus tropi-
dorhynchus Gray (1839) from the United States.
Journal of Mammalogy 78:103-105.

Gager, Y., E. Tarland, D. Lieckfeldt, M. Menage, F. Botero-
Castro, S. J. Rossiter, R. H. S. Kraus, A. Ludwig,
and D. K. N. Dechmann. 2016. The value of mo¬
lecular vs. morphometric and acoustic information
for species identification using sympatric molossid
bats. PLoS ONE 11: e0150780. doi: 10.1371/jour¬
nal.pone.0150780.

Gannon, M. R., A. Kurta, A. Rodriguez-Duran, and M. R.
Willig. 2005. Bats of Puerto Rico: An island focus
and a Caribbean perspective. Texas Tech University
Press. 239 pp.

Genoways, H. H., R. C. Dowler, and C. H. Carter. 1981.
Intraisland and interisland variation in Antil¬
lean populations of Molossus molossus (Mam¬
malia: Molossidae). Annals of Carnegie Museum
50:475^192.

Genoways, H. H., R. J. Baker, J. W. Bickham, and C. J. Phil¬
lips. 2005. Bats of Jamaica. Special Publications,
Museum of Texas Tech University 48:1-155.

Glass, B. P. 1958. Returns of Mexican freetail bats banded
in Oklahoma. Journal of Mammalogy 39:435-437.

Gonzalez-Ruiz, N., J. Ramirez-Pulido, and J. Arroyo-
Cabrales. 2011. A new species of mastiff bat
(Chiroptera: Molossidae: Molossus) from Mexico.
Mammalian Biology 76:461^169.

Griffiths, T. A., and D. Klingener. 1988. On the distribution
of Greater Antillean bats. Biotropica 20:240-251.

Hajibabaei, M., G. A. Singer, P. D. Hebert, and D. A. Hickey.
2007. DNA barcoding: How it complements tax¬
onomy, molecular phylogenetics, and population
genetics. TRENDS in Genetics 23:167-172.

Harris, D., D. S. Rogers, and J. Sullivan. 2000. Phylogeog-
raphy of Peromyscus furvus (Rodentia; Muridae)
based on cytochrome b sequence data. Molecular
Ecology 9:2129-2135.

Husson, A. M. 1962. The bats of Suriname. Zoologische
Verhandelingen, Leiden 58:1-282 + 30 pis.

Larsen, P. A., S. R. Hoofer, M. C. Bozeman, S. C. Pedersen,
H. H. Genoways, C. J. Phillips, D. E. Pumo, and R.
J. Baker. 2007. Phylogenetics and phylogeography
of the Artibeus jamaicensis complex based on
cytochrome-/) DNA sequences. Journal of Mam¬
malogy 88:712-727.

Lindsey and Ammerman—Genetic Diversification of Molossus molossus

13

McDonough, M. M., A.W. Ferguson, L. K. Ammerman, C.
Granjo-Vizcaino, S. F. Burneo, and R. J. Baker.
2011. Molecular verification of bat species col¬
lected in Ecuador: Results of a country-wide
survey. Occasional Papers, Museum of Texas Tech
University 301:1-28.

Miller, G. S. 1904. Notes on the bats collected by William
Palmer in Cuba. Proceedings of the U.S. National
Museum 27:337-348.

Miller, G. S. 1913. Notes on the bats of the genus Molos¬
sus. Proceedings of the U.S. National Museum
46:85-92.

Pallas, P. S. 1766. Miscellanea zoological quibus novae
imprimis atque obscurae animalium species de-
scribunter et observationibus inconibusque illus-
tratum. Hague Comitum. Xii+l-224pp.+ i-xiv pis.

Palumbi, S. R. 1996. Nucleic acids II: The polymerase chain
reaction. Pp. 207-247 in Molecular Systematics. 2 nd
ed. (D. M. Hillis, C. Moritz, and B. K. Mable, eds.).
Sinauer Associates, Inc., Publishers, Sunderland,
Massachusetts.

Piaggio, A. J., E. W. Valdez, M. A. Bogan, and G. S. Spicer.
2002. Systematics of Myotis occultus (Chiroptera:
Vespertilionidae) inferred from sequences of two
mitochondrial genes. Journal of Mammalogy
83:386-395.

Ronquist, F. and J. P. Huelsenbeck. 2003. MrBayes 3: Bayes¬
ian phylogenetic inference under mixed models.
Bioinformatics 19:1572-1574.

Russell, A. L., R. A. Medellin, and G. F. McCracken. 2005.
Genetic variation and migration in the Mexican
free-tailed bat ( Tadarida brasiliensis mexicana).
Molecular Ecology 10:1-16.

Silva-Taboada, G. S. 1979. Los murcielagos de Cuba. Edito-
ria de la Academia de Ciencias de Cuba. Havanna,
Cuba.

Simmons, N. B. 2005. Order Chiroptera. Pp. 312-529 in
Mammal Species of the World: A taxonomic and
geographic reference. 3 rd ed. (D. E. Wilson and D.

Addresses of authors:

Laramie L. Lindsey

Department of Biological Sciences
Texas Tech University
Lubbock, TX 79409 USA
laramie. lindsey@ttu. edu

M. Reeder, eds). John Hopkins University Press,
Baltimore, Maryland.

Smith, M. F., and J. L. Patton. 1991. Variation in mitochon¬
drial cytochrome b sequence in natural populations
of South American akodontine rodents (Muridae:
Sigmodontinae). Molecular Biology and Evolution
8:85-103.

Sudman, P. D., L. J. Barkley, and M. S. Hafner. 1994. Familial
affinity of Tomopeas ravus (Chiroptera) based on
protein electrophoretic and cytochrome b sequence
data. Journal of Mammalogy 75:365-377.

Sun, K-P, J. Feng, T-L. Jiang, J. Ma, Z-Z. Zhang, and L-R.
Jin. 2008. A new cryptic species of Rhinolophus
macrotis (Chiroptera: Rhinolophidae) from Jiangxi
Province, China. Acta Chiropterologica 10:1-10.

Sun, H, J. Dong, H. Shi, M. Ren, and P. Hua. 2015. The
complete mitochondrial genome of a Chinese ru¬
fous horseshoe bat subspecies, Rhinolophus sinicus
sinicus (Chiroptera: Rhinolophidae). Mitochondrial
DNA 5:1-2.

Tamura, K., D. Peterson, N. Peterson, G. Stetcher, M.
Nei, and S. Kumar. 2011. MEGA5: Molecular
evolutionary genetics analysis using maximum
likelihood, evolutionary distance, and maximum
parsimony methods. Molecular Biology and Evolu¬
tion 28:2731-2739.

Vaughan, T. A. 1978. Mammalogy. W. B. Saunders, Phila¬
delphia. x+522 pp.

Warner, J. W., J. L. Patton, A. L. Gardner, and R. J. Baker.

1974. Karyotypic analyses of twenty-one species of
molossid bats (Molossidae: Chiroptera). Canadian
Journal of Genetics and Cytology 16:165-176.

Whitman, D. W., and A. A. Agrawal. 2009. What is pheno¬
typic plasticity and why is it important? Pp. 1-63
in Phenotypic plasticity of insects: Mechanisms
and consequences. (D. W. Whitman and T. N. An-
anthakrishnan, eds.). Science Publishers, Enfield,
New Hampshire.

Loren K. Ammerman

Department of Biology
Angelo State University
San Angelo, TX 76909 USA
loren. ammerman@angelo. edu

14

Occasional Papers, Museum of Texas Tech University

Appendix

Species, catalog number, tissue number, locality, and GenBank accession numbers generated in the cytb and
analysis. ASK (tissue number), FN (tissue number), QCAZ (catalog number), and ASNHC (catalog number) = Angelo
State Natural History Collection, Angelo State University; TK (tissue number) and TTU (catalog number) = Natural
Science Research Laboratory, Museum of Texas Tech University; CM (catalog number) = Carnegie Museum; USNM
(catalog number) = United States National Museum; BDP (tissue number) and FMNH (catalog number) = Field Mu¬
seum of Natural History; KM = GenBank accession number.

Molossus alvarezi (2).—MEXICO: Yucatan; Tekax; Merida, Colegio Peninsular ASNHC7023/FN32915/
KM3 87333; A SNHC7024/FN32916/KM3 87334.

Molossus bondae (1).—JAMAICA: St. Catherine Parish; 0.2 mi E Watermount CM44668/TK9218/KM387368.

Molossus rufus (4).—PARAGUAY: San Pedro; Yaguarete Forests; 1.7 km E Headquarters TTU96091/TK56709/
KM387361. GUYANA: Upper Demerara-Berbice; Dubulay Ranch, Region 10, Subregion 2 USNM582416/TK86608/
KM387345; USNM582418/TK86648/KM387380; USNM582419/TK86649/KM387346.

Molossus coibensis (2).—VENEZUELA: Bolivar; 12 km S El Manteco CM78716/TK19168/KM387358;
CM78717/TK19170/KM387322.

Molossus molossus (5).—BRAZIL: Sao Paulo; Esta^ao Biologica de Boraceia FMNH219980/BDP3265/
KM387326; BDP3271/KM387327; BDP3280/KM387328. EL SALVADOR: La Paz; Playa El Zapote TTU60988/
TK34864/KM387373; TTU60989/TK34865/KM387377.

Molossus molossus daulensis (6).—ECUADOR: El Oro; Manchala; Cuidad Manchala, Junin St., Hotel Mercy
ASNHC 14120/ASK7779/KM387366; ASNHC14121/ASK7786/KM387356; QCAZ8620/ASK7787/KM058059.
ECUADOR: El Oro; Palmales, Reserva Militar Arenillas TTU102336/TK135112/KM387375. ECUADOR: Guayas;
Manglares Churute; Guardiania Del Parque TTU103736/TK134642/KM387351. ECUADOR: Guayas; Bosque Pro¬
tector Cerro Blanco, Centro de Visitantes TTU103300/TK134887/KM387363.

Molossus molossus molossus (39).—BARBADOS: St. Thomas Parish; Welchman Hall Gully, 0.5 km N Welch¬
man Hall TTU109911/TK 151406/KM387364. BARBADOS: Christ Church Parish; Graeme Hall Swamp, 0.5 kmN
St. Lawrence TTU109888/TK151439/KM387353. BOLIVIA: La Paz; 1 mi W Puerto Linares TTU34957/TK14589/
KM387336. ECUADOR: Zamora-Chinchipe; 1 km N, 0.8 km E Zamora ASNHC14140/ASK7760/KM387365;
QCAZ8592/ASK7759/KM387325. ECUADOR: Morona-Santiago; north of Macas, Nueva Jerusalem ASNHC 14133/
ASK7730/KM387324. ECUADOR: Orellana; Estacion Cientifica Yasuni FMNH213845/BDP5170/KM387367;
FMNH213847/BDP5153/KM387331; FMNH213850/BDP5175/KM387332. GRENADA: St. George; CheminRiver,
0.5 km E Confer CM63415/TK18548/KM387337; CM63432/TK18565/KM387338. GRENADINES: Union Island;
Big Sand Beach, 1 km N Clifton CM63270/TK18622/KM387370. GRENADINES: Union Island; 0.5 km N Clifton
CM63488/TK18651/KM387371. GUYANA: Upper Demerara-Berbice; Dubulay Ranch USNM582412/TK86625/
KM387344; TK86651/KM387362. GUYANA: Upper Demerara-Berbice; Dubulay Ranch, Region 10, Subregion 2
USNM582423/TK86602/KM387341; USNM582424/TK86603/KM387360; USNM582425/TK86604/KM387342;
USNM582426/TK86605/KM387379; USNM582361/TK86607/KM387343; USNM582428/TK86653/KM387347;
USNM582429/TK86654/KM387348; USNM582430/TK86655/KM387349. PARAGUAY: Pte. Hayes; EstanciaLoma
PoraTTU80400/TK64488/KM387323. PARAGUAY: Cordillera; Estancia Sombrero TTU80302/TK64799/KM387378.
PUERTO RICO: Vieques Island; Green Beach Gate FMNH206530/BDP4903/KM387376. PUERTO RICO: Vieques
Island; Ammunition Bunkers FMNH206531/BDP4906/KM387329; FMNH206532/BDP4907/KM387330. ST. VIN¬
CENT AND THE GRENADINES: Bequia; 2.3 km NE Port Elizabeth TTU105217/TK128560/KM387350;TTU105218/
TK128561/KM387374. SURINAME: Paramaribo; Paramaribo CM64421/TK11333/KM387335; CM64432/TK11344/
KM387357. SURINAME: Saramacca; Raleigh Falls TTU35731/TK17231/KM114224; TTU35732/TK17232/

Lindsey and Ammerman—Genetic Diversification of Molossus molossus

15

Appendix (cont.)

KM387369. UNITED STATES: St. Croix; West End Quarter; Brugall Rum Factory; 0.45 km E, 0.9 km N Frederiksted
TTU111461/TK148718/KM387381. UNITED STATES: St. Croix; West End Quarter; Estate Jolly Hill; 0.35 km E, 0.25
km S Jolly Hill TTU111464/TK148739/KM387352. ST. LUCIA; Castries; Union Nature Trail, 0.6 km N, 0.5 km W
TTU109924/TK151281 /KM387382. ST. LUCIA: Dennery; Dennery River, 0.25 km S, 2 km WDennery TTU109943/
TK161331/KM387355. ST. LUCIA: Micoud; Troumassee River, 1.3 kmWMicoud TTU109945/TK161199/KM387354.

Molossus molossus tropidorhynchus (4).—CUBA: Guantanamo Province; Guantanamo Bay Naval Station
TTU52669/TK32043/KM387339; TTU52666/TK32081/KM387359; TTU52648/TK32141/KM387340; TTU52649/
TK32142/KM387372.

Publications of the Museum of Texas Tech University

This publication is available free of charge in PDF format from the website of the Natural Science Research Labo¬
ratory, Museum of Texas Tech University (nsrl.ttu.edu). The authors and the Museum of Texas Tech University
hereby grant permission to interested parties to download or print this publication for personal or educational
(not for profit) use. Re-publication of any part of this paper in other works is not permitted without prior written
permission of the Museum of Texas Tech University.

Institutional subscriptions to Occasional Papers are available through the Museum of Texas Tech University, attn: NSRL
Publications Secretary, Box 43191, Lubbock, TX 79409-3191. Individuals may also purchase separate numbers
of the Occasional Papers directly from the Museum of Texas Tech University.

Series Editor: Robert D. Bradley
Production Editor: Lisa Bradley

— museum

texas tech universil

ISSN 0149-175X

Museum of Texas Tech University, Lubbock, TX 79409-3191