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Research Article
The taxonomic status of Myotis nesopolus larensis (Chiroptera, Vespertilionidae) and new insights on the diversity of Caribbean Myotis
expand article infoRoberto Leonan M. Novaes‡, Vinícius C. Cláudio‡§, Roxanne J. Larsen|, Don E. Wilson§, Marcelo Weksler‡, Ricardo Moratelli
‡ Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
§ Smithsonian Institution, National Museum of Natural History, Washington DC, United States of America
| University of Minnesota, Saint Paul, United States of America
¶ Fundação Oswaldo Cruz, Fiocruz Mata Atlântica, Rio de Janeiro, Brazil

Corresponding author: Roberto Leonan M. Novaes ( robertoleonan@gmail.com )

Academic editor: DeeAnn Reeder
© 2021 Roberto Leonan M. Novaes, Vinícius C. Cláudio, Roxanne J. Larsen, Don E. Wilson, Marcelo Weksler, Ricardo Moratelli.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation: Novaes RLM, Cláudio VC, Larsen RJ, Wilson DE, Weksler M, Moratelli R (2021) The taxonomic status of Myotis nesopolus larensis (Chiroptera, Vespertilionidae) and new insights on the diversity of Caribbean Myotis. ZooKeys 1015: 145-167. https://doi.org/10.3897/zookeys.1015.59248
ZooBank: urn:lsid:zoobank.org:pub:F2612906-AA93-4B26-B42F-8477F046EB27
Open Access

Abstract

Myotis nesopolus currently comprises two subspecies. The nominate subspecies (M. n. nesopolus) occurs on the Caribbean islands of Curaçao and Bonaire, Netherlands Antilles, whereas M. n. larensis is known from mainland South America in northeastern Colombia and northwestern Venezuela. Our Maximum Likelihood phylogenetic analyses of cytochrome-b gene sequences recovered M. nesopolus as a paraphyletic group, with M. n. nesopolus and M. n. larensis as non-sister lineages. The haplotype network indicates that these two subspecies do not share any haplotypes and are in different evolutionary trajectories. Additionally, these two subspecies can be distinguished on the basis of qualitative and quantitative morphological traits. This pattern supports the recognition of M. nesopolus and M. larensis as full species. Our results also reveal that the assemblage of Caribbean Myotis do not form a monophyletic group. Caribbean species are phylogenetically close to mainland species from northern South America and Central America, suggesting that colonization of Caribbean islands happened multiple times.

Resumo

Atualmente Myotis nesopolus compreende duas subespécies: M. n. nesopolus ocorre nas ilhas caribenhas de Curaçao e Bonaire, Antilhas Holandesas, enquanto M. n. larensis é conhecido para o continente da América do Sul, no nordeste da Colômbia e noroeste da Venezuela. Nossa inferência filogenética por Máxima Verossimilhança recuperou M. nesopolus como parafilética, com M. n. nesopolus e M. n. larensis sendo linhagens não-irmãs. Além disso, essas duas subespécies não compartilham nenhum haplótipo. Adicionalmente, as subespécies podem ser diferenciadas a partir de caracteres morfológicos e morfométricos. Esse achado suporta o reconhecimento de M. nesopolus e M. larensis como espécies distintas. Nossos resultados revelam que os Myotis do Caribe não formam um grupo monofilético. Espécies caribenhas são filogeneticamente próximas de espécies continentais das Américas Central e do Sul, sugerindo que a colonização das ilhas do Caribe aconteceu por múltiplos eventos de dispersão.

Keywords

Bats, biogeography, Lesser Antilles, morphology, morphometry, taxonomy, South America, Venezuela

Introduction

Myotis Kaup, 1829 (Vespertilionidae, Myotinae) comprises more than 120 species distributed worldwide, and is the most speciose genus of bats (Simmons 2005; Burgin et al. 2018). Twenty-seven species are recognized from the Neotropics (Wilson 2008; Moratelli et al. 2017, 2019a; Carrión-Bonilla and Cook 2020). However, molecular evidence has revealed that the current species richness is underestimated (Clare et al. 2011; Larsen et al. 2012a; Chaverri et al. 2016; Moratelli et al. 2017).

Two subspecies of Myotis nesopolus Miller, 1900 are recognized. The nominate subspecies, M. n. nesopolus, is known from Curaçao and Bonaire in the Netherlands Antilles. The other subspecies, M. n. larensis LaVal, 1973, is known from mainland South America in northeastern Colombia and northwestern Venezuela (LaVal 1973; Wilson 2008; Muñoz-Garay and Mantilla-Meluk 2012; Moratelli et al. 2013). LaVal (1973) described Myotis larensis as a full species from “Río Tocuyo, Lara, Venezuela”. Genoways and Williams (1979), however, treat larensis as a subspecies of Myotis nesopolus. Miller’s (1900) description of M. nesopolus was based on one specimen from Willemstad, Curaçao, Netherlands Antilles. Subsequently, Genoways and Williams (1979) considered that representatives of Myotis from Bonaire island, originally identified as Myotis nigricans (Schinz, 1821), were misidentifications of M. nesopolus, which was confirmed by Moratelli et al. (2017).

Previous molecular and morphological studies questioned the subspecific status of mainland populations of M. nesopolus, suggesting that the two subspecies might represent different species (Larsen et al. 2012b; Moratelli et al. 2013, 2017). Here we reassess the taxonomic status of M. n. larensis in the light of new morphological and genetic analyses.

Materials and methods

Specimens examined

Specimens of M. nesopolus used in this study are deposited in the American Museum of Natural History (AMNH, New York, USA), Carnegie Museum of Natural History (CM, Pittsburgh, USA), Smithsonian’s National Museum of Natural History (USNM, Washington DC, USA), and Museum of Texas Tech University (TTU, Lubbock, USA). We examined the holotype of M. n. nesopolus (USNM 101849), two topotypes from Curaçao (CM 52432, USNM 105128), and nine specimens from Bonaire (Appendix 1). Material of M. n. larensis includes the holotype (AMNH 130709), and fifteen additional specimens from mainland Venezuela.

Molecular analyses

Phylogenetic analyses of complete cytochrome-b gene (cyt-b, 1,140 bp, no gaps) sequences were conducted for the Neotropical assemblage of Myotis. A total of 122 sequences, including outgroups, were retrieved from GenBank (Appendix 2). We used the palearctic species Myotis brandtii (Eversmann, 1845) and Myotis gracilis Ognev, 1927 as outgroups because they are sister to the Neotropical clade (see Ruedi et al. 2013). Multiple sequence alignment of full length cyt-b sequences were performed with MEGA X (Kumar et al. 2018), using MUSCLE algorithm with default settings (Edgar 2004). Subsequently, the Bayesian Information Criterion (BIC), as implemented in JModelTest2 (Darriba et al. 2012), was used to determine the best-fit models of nucleotide substitution. The Hasegawa-Kishino-Yano model (Hasegawa et al. 1985) was chosen to correct the heterogeneity rate using gamma-distribution with invariant sites (i.e., HKY + Γ + I).

The phylogenetic analysis was carried out using Maximum Likelihood (ML) method (Felsenstein 1981), in the software RAxML v8.0 (Stamatakis 2014). To assess the nodal support, we calculated a nonparametric bootstrap using 1000 replications. Genetic distance values for cyt-b sequences were calculated in MEGA X using the Kimura 2-parameter model (Kimura 1980).

To understand the population structure of M. n. nesopolus, M. n. larensis and other phylogenetically related population groups, we built a haplotype network (distribution of haplotypes by previously defined population groups) using the median-joining algorithm in the Network 4.6.1.3 software (Bandelt et al. 1999).

Morphological and morphometric analyses

We examined 284 specimens for the morphological comparisons, including M. n. nesopolus (N = 10), M. n. larensis (N = 9) and 14 species of Neotropical Myotis deposited in 11 collections in Brazil, Canada and United States (Appendix 1). Specimens were identified following Wilson (2008) and Moratelli et al. (2011, 2013, 2017). The main qualitative morphological characters used in the comparisons were: (i) presence and height of sagittal crest; (ii) presence and height of lambdoidal crests; (iii) inclination shape of the frontal and parietal bones; (iv) presence of a fringe of hairs along the trailing edge of the uropatagium; (v) dorsal and ventral fur texture and height; (vi) pattern of fur coloring, with the capitalized color nomenclature following Ridgway (1912).

We took one external and 16 craniodental measurements (Table 1), using digital calipers to the nearest 0.01 mm. Measurements were made under binocular microscopes with low magnification (usually 6×). Measurements were recorded from adults and are reported in millimeters (mm). The length of ear and body mass were recorded from skin labels. We used a principal component analysis (PCA) to identify general trends of cranial size and shape variation among samples, and a discriminant function analysis (DFA), with a priori identification of samples, to compare skull size and shape of M. n. nesopolus (N = 9) and M. n. larensis (N = 9). For these analyses, we selected a subset of 11 craniodental dimensions representing different axes of the length and width of skull, rostrum, and mandible, as follows: greatest length of skull, including incisors (GLS), condylo-incisive length (CIL), mastoid breadth (MAB), braincase breadth (BCB), interorbital breadth (IOB), postorbital breadth (POB), breadth across canines (BAC), breadth across molars (BAM), maxillary toothrow length (MTL), molariform toothrow length (M1–M3), and mandibular toothrow length (MAN). PCA and DFA analyses were run in R software (R Development Core Team 2012) using the MASS and Lattice packages (Venables and Ripley 2002; Sarkar 2008). Because multivariate procedures require complete data sets, missing values (ca 1.5% of the total dataset) were estimated from the existing raw data using the Amelia II package (Honaker et al. 2011) implemented in R software. Measurements were transformed to natural logs and covariance matrices were computed considering all variables. Subsequently, an analysis of variance using Mann-Whitney statistics was employed to test whether the population samples differ in cranial dimensions. The comparison was made using p-values and when less than 0.001 were considered as statistically significant. This analysis was run in the software PAST 3.3 (Hammer et al. 2001).

Table 1.
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Description of cranial, mandibular, and external dimensions (and their abbreviations). Lengths were measured from the anteriormost point or surface of the 1st structure to the posteriormost point or surface of the 2nd structure, except as specified.

Measurements Acronyms Descriptions
Forearm length FA From the elbow to the distal end of the forearm including carpals
Greatest length of skull GLS From the apex of the upper internal incisors, to the occiput
Condylo-canine length CCL From the anterior surface of the upper canines to a line connecting the occipital condyles
Condylo-basal length CBL From the premaxillae to a line connecting the occipital condyles
Condylo-incisive length CIL From the apex of upper internal incisors to a line connecting the occipital condyles
Basal length BAL Least distance from the apex of upper internal incisors to the ventral margin of the foramen magnum
Zygomatic breadth ZYG Greatest breadth across the outer margins of the zygomatic arches
Mastoid breadth MAB Greatest breadth across the mastoid region
Braincase breadth BCB Greatest breadth of the globular part of the braincase
Interorbital breadth IOB Least breadth between the orbits
Postorbital breadth POB Least breadth across frontals posterior to the postorbital bulges
Breadth across canines BAC Greatest breadth across outer edges of the crowns of upper canines, including cingulae
Breadth across molars BAM Greatest breadth across outer edges of the crowns of upper molars
Maxillary toothrow length MTL From the upper canine to M3
Molariform toothrow length M1–M3 From M1 to M3
Mandibular length MAL From the mandibular symphysis to the condyloid process
Mandibular toothrow length MAN From the lower canine to m3

Results

Molecular analyses

The ML phylogeny based on cyt-b sequences indicates that M. nesopolus, as currently recognized, is paraphyletic, with M. n. nesopolus more closely related to an eastern Peruvian unidentified lineage, whereas M. n. larensis was recovered more closely related to an unidentified lineage from western Ecuador (Fig. 1), although this phylogeny and branching events has low nodal support. These unidentified species from Peru and Ecuador were originally designated as Myotis nigricans by the original collector due to morphological similarities. However, M. nigricans has been recovered as polyphyletic and considered a cryptic species complex in many studies (Moratelli et al. 2011, 2013, 2016, 2017; Larsen et al. 2012a). Therefore, we decided not to give a name to the lineages related to M. nesopolus and M. larensis. We emphasize that the previous identification of these specimens as M. nigricans by one of our authors (RJL) in a previous study (Larsen et al. 2012a) indicates that these populations are morphologically distinct from those considered here as M. nesopolus and M. larensis.

The Caribbean Myotis species do not form a monophyletic group, being related to Myotis atacamensis (Lataste, 1892) and other mainland putative species. Nevertheless, the phylogenetic relationship of Caribbean Myotis clade is not fully resolved, since a polytomy was recovered among M. sp. 3 from Honduras and the ancestral lineage of M. n. nesopolus and M. sp. 2 from Peru, and of M. n. larensis and M. sp. 1 from Ecuador. Similarly, a polytomy was recovered among M. atacamensis, M. martiniquensis and an ancestral lineage of M. dominicensis, M. nyctor and M. sp. 4 from Suriname (Fig. 1).

Figure 1. 

Phylogenetic tree resulting from the Maximum Likelihood analysis of cytochrome-b sequences of species of Myotis. Nodal support was calculated by bootstrap and black solid circles are values between 100–95% and hollow white circle are values between 94–90%. Values less than 90% were not indicated. The rectangle encloses the phylogenetic relationship, where branches were transformed to cladogram, among M. nesopolus, M. larensis, Caribbean Myotis (colored terminals) and mainland haplogroups of five more closely related species and candidate species.

The average cyt-b pairwise distance between M. n. larensis and Myotis sp. 1 from western Ecuador is 2.1% ± 0.3; between M. n. nesopolus and Myotis sp. 2 from eastern Peru is 3.8% ± 0.4; and between M. n. nesopolus and M. n. larensis is 4.0% ± 0.3 (Table 2). Levels of intraspecific variation were less than 0.8% for all recognized and putative species (Table 2).

Table 2.
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Average Kimura 2-parameter genetic distances within (along diagonal) and among (below diagonal) Myotis taxa based on cytochrome-b gene sequences. Boldface value indicates the distance between M. larensis and M. nesopolus. Hyphen indicates groups with a single sequence.

Taxa 1 2 3 4 5 6 7 8 9 10 11 12
1 M. atacamensis (Peru) –
2 Myotis sp. 4 (Suriname) 0.085 0.002
3 M. nyctor (Grenada) 0.103 0.080 –
4 M. nyctor (Barbados) 0.089 0.070 0.002 0.004
5 M. dominicensis (Dominica) 0.080 0.087 0.092 0.088 0.001
6 M. martiniquensis (Martinique) 0.087 0.093 0.089 0.094 0.887 0.002
7 M. n. larensis (Venezuela) 0.093 0.107 0.127 0.119 0.097 0.096 0.003
8 Myotis sp. 1 (W Ecuador) 0.091 0.104 0.134 0.120 0.092 0.093 0.021 0.002
9 Myotis sp. 2 (E Peru) 0.104 0.115 0.138 0.126 0.107 0.104 0.034 0.033 0.001
10 M. n. nesopolus (Bonaire) 0.103 0.115 0.147 0.124 0.104 0.106 0.040 0.044 0.038 0.008
11 Myotis sp. 3 (Honduras) 0.103 0.116 0.133 0.120 0.107 0.105 0.046 0.049 0.056 0.053 –
12 M. attenboroughi (Tobago) 0.081 0.093 0.101 0.099 0.091 0.088 0.068 0.075 0.076 0.078 0.079 0.000

The haplotype network indicates that there are no haplotypes shared between M. n. nesopolus, M. n. larensis, and phylogenetically close species (Fig. 2). The haplotypes were grouped into small clusters well-distributed among the populations, with no central haplotype. The network indicates spatial structuring with isolation among the population groups tested, agreeing with what was obtained by phylogenetic inference.

Figure 2. 

Haplotype network from cyt-b sequences of Myotis nesopolus (blue), Myotis larensis (red) and other mainland closest Myotis lineages from Central and South America. Each tick mark represents a single base-pair mutation.

Morphological analyses

The first principal component (PC1) accounted for 87% of the total craniometric variation, and represents overall skull size (Fig. 3A, B). Along this axis, scores of M. n. larensis and M. n. nesopolus do not overlap. On the other hand, the two samples overlap broadly along the second principal component (PC2 = 5%) which represents overall skull shape. The distribution of M. n. larensis and M. n. nesopolus samples across size and shape axes in the discriminant analysis (Fig. 3C, D) is similar to that observed in the PCA. Measurements associated with skull and mandible length (GLS, CIL, MAN) and skull width (IOB) were the most useful to discriminate samples (Table 3). Considering that skull axes are represented by the set of measurements used in the morphometric multivariate analysis, these results reveal that M. n. larensis and M. n. nesopolus have distinct skull size and shape.

Figure 3. 

Plots showing convex-hulls and vector correlation of cranial measurements of Principal Component Analysis (A, B) and Discriminant Function Analysis (C, D) for Myotis nesopolus from Curaçao (black square), Myotis nesopolus from Bonaire (blue triangles) and Myotis larensis from Venezuela mainland (red dots).

Table 3.
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Vector correlation loadings with original variables of principal components (PC1 and PC2) and discriminant functions (DF1 and DF2) for selected samples of M. larensis and M. nesopolus. See Table 1 for variable abbreviations.

Measurements PC 1 PC2 DF1 DF2
MAN 0.324 -0.091 0.063 0.016
GLS 0.573 -0.103 0.109 0.026
CIL 0.506 -0.056 0.093 0.027
MAB 0.097 0.327 0.012 0.012
BCB 0.109 0.108 0.019 0.003
IOB 0.258 0.775 0.051 0.014
POB -0.02 0.363 -0.005 0.026
BAC 0.198 0.031 0.04 0.021
BAM 0.277 -0.165 0.059 -0.015
MTL 0.262 -0.088 0.052 0.011
M1–3 0.187 -0.298 0.040 -0.007

Populations from the Antilles and mainland South America do not overlap in measurements of several characters, which may be useful in distinguishing species: M. n. larensis forearm length ranges from 31.2 to 33.2 mm, and GLS from 13.6 to 14.5 mm; M. n. nesopolus forearm length ranges from 28.2 to 31.0 mm, and GLS from 12.9 to 13.4 mm. The Mann-Whitney test found significant differences in 11 of the 14 measurements tested (Table 4).

Table 4.
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Selected measurements (mm) of M. larensis from Venezuela and M. nesopolus from Curaçao and Bonaire. Descriptive statistics include the mean, range (in parentheses), and sample size. See Table 1 for variable abbreviations. Mann-Whitney Test p-values was used to compare cranial measurements between samples. Measurements with hyphen (–) not were tested due to disparate samples size.

Measurements Myotis larensis Myotis nesopolus P–value
FA 32.2 (31.2–33.2) 7 29.7 (28.2–31.0) 11 –
GLS 13.7 (13.3–14.4) 9 12.9 (12.8–13.1) 9 < 0.001
CCL 12.1 (11.5–12.7) 9 11.6 (11.4–11.8) 9 < 0.001
CBL 12.8 (12.4–13.5) 9 12.2 (12.0–12.5) 9 < 0.001
CIL 12.9 (12.6–13.6) 9 12.4 (12.2–12.6) 9 < 0.001
BAL 11.6 (11.2–12.4) 9 11.1 (10.9–11.3) 9 < 0.001
ZYG 8.1 (8.0–8.2) 3 7.8 (7.7–8.0) 8 –
MAB 5.3 (5.1–5.6) 9 6.7 (6.4–6.8) 9 0.247
BCB 6.2 (6.1–6.3) 9 6.1 (5.9–6.2) 9 0.017
IOB 4.4 (4.0–4.7) 9 4.0 (3.9–4.2) 9 0.003
POB 3.3 (3.2–3.4) 9 3.3 (3.2–3.5) 9 0.374
BAC 3.3 (3.2–3.5) 9 3.0 (3.0–3.2) 9 < 0.001
BAM 5.3 (5.1–5.5) 9 4.9 (4.8–5.0) 9 < 0.001
MTL 5.2 (5.0–5.4) 9 4.8 (4.7–4.9) 9 < 0.001
M1M3 2.9 (2.8–3.2) 9 2.7 (2.6–2.8) 9 < 0.001
MAL 9.8 (9.5–10.3) 4 9.0 (8.8–9.2) 9 –
MAN 5.5 (5.3–5.9) 8 5.1 (4.9–5.3) 9 < 0.001

Population samples from the Antilles and mainland South America have several qualitative morphological differences. Specimens of M. n. nesopolus have moderately silky fur (length of dorsal fur 5–6 mm; length of ventral fur 3–4 mm); dorsal fur Dresden-Brown with little contrast between bases and tips slightly lighter tips; ventral fur with blackish bases and Light-Buff tips (Fig. 4A). Specimens of M. n. larensis have long silky fur (length of dorsal fur 6–8 mm; length of ventral fur 5–6 mm); dorsal fur strongly bicolored, with blackish bases (2/3) and Tawny-Olive tips (1/3); ventral fur with blackish bases and whitish tips (Fig. 4B). The sagittal crest is absent in M. n. nesopolus, the lambdoidal crests are generally absent or very low, and the parietal is inclined forward. Sagittal and lambdoidal crests are present in M. n. larensis, ranging from low to moderate in development, and the parietal is not inclined forward. In both populations, the second upper premolar (P3) is aligned in the toothrow and visible in labial view, and the occipital region is always rounded (Fig. 5).

Figure 4. 

Dorsal (left) and ventral (right) fur of a specimen of Myotis nesopolus (CM 52217 [A]) from Bonaire and the holotype of Myotis larensis (USNM 441737 [B]) from Lara, Venezuela.

The congruence between the molecular and morphological evidence indicates that the two subspecies of M. nesopolus do not form a clade. Thus, M. larensis represents an independent evolutionary lineage and should be treated as a full species.

Figure 5. 

Skull profiles of Myotis larensis (AMNH 130709 [holotype]) from Venezuela in lateral (A), ventral (B) and dorsal (C) views; and Myotis nesopolus (USNM 105128 [topotype]) from Curaçao in lateral (D), ventral (E) and dorsal (C) views. The image of the M. nesopolus skull was inverted.

Description and comparisons

Myotis larensis is a small-sized bat (total length 78–82 mm; forearm length 31.2–33.2; body mass 3–5 g), morphologically similar to several Neotropical congeners. Ears are moderate in size (length 10–13 mm), and when laid forward extend halfway from eye to nostril. Antitragal notch is barely evident. Membranes are Mummy-brown. Fur on dorsal surface of uropatagium extends slightly past the knees. Plagiopatagium is attached to the foot at toes level by a broad band of membrane. Third metacarpal, tibia, and skull are long in relation to forearm (mean ratios 0.96, 0.48, and 0.43, respectively; see LaVal (1973)).

Myotis larensis can be distinguished from all Caribbean and South American congeners by qualitative and quantitative traits. It differs from M. nesopolus by its larger size (no overlapping in forearm length and greatest length of skull), presence of sagittal crest, and dorsal fur longer and strongly bicolored. Considering the Myotis species that occurs in the northern South America, M. larensis differs from M. albescens (É. Geoffroy, 1806) by the absence of a fringe of hairs along the trailing edge of the uropatagium; from M. keaysi J. A. Allen, 1914, M. pilosatibialis LaVal, 1973, M. riparius Handley, 1960, and M. simus Thomas, 1901 by the long silky dorsal fur strongly bicolored. Myotis larensis can also be distinguished from M. simus by the plagiopatagium broadly attached at base of the toes. Myotis larensis differs from M. diminutus Moratelli & Wilson, 2011 by its larger cranial dimensions and dorsal fur strongly contrasting; from M. handleyi Moratelli et al., 2013 by its strongly contrasting and long silk dorsal fur and shorter forearm; from M. oxyotus (Peters, 1867) by having a smaller skull, less steeply sloping frontals and strongly contrasting dorsal fur. Myotis larensis differs from M. attenboroughi Moratelli et al., 2017 by its lighter and strongly contrasting dorsal fur and larger skull; and from M. clydejonesi Moratelli et al., 2016 by its moderate steeply sloping frontals, less inflated braincase, smaller skull and dorsal fur strongly contrasting. Myotis larensis differs from M. caucensis Allen, 1914 by its smaller skull and strongly contrasting dorsal fur. Myotis larensis can be distinguished from M. cf. nigricans from northern South America (sensu Moratelli et al. 2013) by the lighter dorsal and ventral fur, more developed sagittal and lambdoid crests and parietal not inclined forward.

Discussion

Genoways and Williams (1979) determined that mainland and island specimens of M. larensis and M. nesopolus, respectively, were morphometrically similar, with Venezuelan specimens slightly smaller than those from Curaçao. As a result, they recognized M. larensis as a subspecies of M. nesopolus, which was followed by subsequent authors (e.g., Simmons 2005; Wilson 2008; Moratelli et al. 2019b). However, our results do not support this arrangement, indicating a morphometric discontinuity and qualitative morphological differences between M. larensis and M. nesopolus.

Previous phylogenetic studies based on mitochondrial and nuclear DNA recovered M. nesopolus and M. larensis as sister lineages and questioned the subspecific status of M. larensis because the cyt-b genetic distance of 4% between mainland and Antilles populations suggests a potential for separation at the species level (see Bradley and Baker 2001; Larsen et al. 2012b). However, this study did not include the mainland samples from Ecuador and Peru. Our phylogenetic analyses revealed that M. nesopolus and M. larensis are not sister lineages and do not share haplotypes. The genetic distances between M. nesopolus, M. larensis and their sister species are greater than 2%. About this, Bradley and Baker (2001) indicate that genetic distance values between 2 and 11% from cyt-b sequences had a high probability of being indicative of conspecific populations or valid species and merit additional study concerning specific status. Our investigation found a conspicuous phenotypic discontinuity in variation of both the size and shape of the skull and other external characters. Thus, the strong congruence between the morphological, morphometric and molecular evidence presented here supports the hypothesis that M. larensis represents a full species.

Nevertheless, it is important to mention the limitation of cyt-b gene for establishing species boundaries in the Caribbean clade, particularly between M. larensis and M. sp. 1 from Ecuador and between M. nesopolus and M. sp. 2 from Peru. Although widely used (e.g., Larsen et al. 2012a, b; Moratelli et al. 2016, 2017; Carrión-Bonilla and Cook 2020), the application of cyt-b data to species delimitation and inference of phylogenetic relationships in Myotis from the Caribbean clade was insufficient. This demonstrates the need to expand the use of new genetic markers for future systematic studies with the Caribbean Myotis assemblage.

With the recognition of M. larensis at the species level hierarchy, M. nesopolus is restricted to Bonaire and Curaçao and is the only species of the genus found in these islands (Fig. 6). Similarly, other Caribbean islands have unique Myotis species, including: Myotis dominicensis Miller, 1902 restricted to Dominica and Guadeloupe; Myotis martiniquensis LaVal, 1973 is restricted to Martinique; Myotis attenboroughi is restricted to Tobago; and Myotis nyctor LaVal & Schwartz, 1974 is restricted to Barbados and Grenada (LaVal 1973; Larsen et al. 2012a; Moratelli et al. 2017). However, the taxonomic status of some populations of these species needs to be reassessed. For example, Myotis nyctor was described from Barbados and subsequently recorded from Grenada (LaVal 1973; LaVal and Schwartz 1974; Moratelli et al. 2017). Although our phylogenetic analysis grouped the samples of M. nyctor from Barbados (N = 5) and Grenada (N = 1) in the same clade (Fig. 1), and with low genetic distance between them (ca 0.2%; Table 2), there are qualitative and quantitative morphological differences between specimens from these two islands (see Larsen et al. 2012a). The similarity in the cyt-b sequences between Grenada and Barbados specimens may be explained by the retained ancestral polymorphism due to the very recent separation (Stadelmann et al. 2007; Larsen et al. 2012a).

Figure 6. 

Geographic distributions of Myotis larensis (restricted to mainland South America in Venezuela and Colombia) and Caribbean Myotis species M. nesopolus, M. dominicensis, M. martiniquensis, M. nyctor, and M. attenboroughi.

The biogeographic interpretations made by Larsen et al. (2012b) suggest at least two independent Myotis invasions into the Lesser Antilles, and reverse colonization by Caribbean Myotis to mainland Central and South America—the latter being a well-documented pattern in other Caribbean bat lineages (Dávalos 2005, 2006, 2010; Genoways et al. 2005; Pavan and Marroig 2017; Tavares et al. 2018). In addition, some biogeographic and ecological aspects suggest the need for taxonomic revision of some species. The distance and geographic isolation between Barbados and Grenada (ca 255 km) are greater than between Dominica and Martinique (ca 42 km), each one having a unique Myotis species. Moreover, Barbados and Grenada are separated by the Tobago Basin, with an ocean depth of approximately 2500 m and no ridges that may have connected these two populations during glaciation periods (Speed 1981; Humphrey 1997; Graham 2003). Considering the apparent low vagility and the small home range of Myotis in general (e.g., LaVal and Fitch 1977; Castella et al. 2001; Moratelli et al. 2019b), it is possible that the populations of M. nyctor from these two islands are isolated and on different evolutionary trajectories. The same rationale might be valid for M. dominicensis, where the populations from Guadeloupe and Martinique are isolated by approximately 42 km of sea. However, there are several oceanic ridges between these two islands, which may have served as bridges connecting these two populations during the last glaciation (Speed 1981; Humphrey 1997; Graham 2003). Thus, we suggest that future studies on systematics and biogeography of Caribbean Myotis should focus on the definition of the taxonomic status of island populations from Grenada and Guadeloupe.

With the recognition of M. larensis as a full species, 28 species of Neotropical Myotis (sensu Stadelmann et al. 2007) are currently recognized: M. albescens (É. Geoffroy, 1806), M. ruber (É. Geoffroy, 1806), M. nigricans (Schinz, 1821), M. levis (I. Geoffroy, 1824), M. chiloensis (Waterhouse, 1840), M. oxyotus (Peters, 1866), M. atacamensis (Lataste, 1892), M. nesopolus Miller, 1900, M. simus Thomas, 1901, M. dinellii Thomas, 1902, M. dominicensis Miller, 1902, M. caucensis Allen, 1914, M. keaysi J.A. Allen, 1914, M. riparius Handley, 1960, M. elegans Hall, 1962, M. larensis LaVal, 1973, M. martiniquensis LaVal, 1973, M. pilosatibialis LaVal, 1973, M. nyctor LaVal & Schwartz, 1974, M. diminutus Moratelli & Wilson, 2011, M. lavali Moratelli et al., 2011, M. izecksohni Moratelli et al., 2011, M. handleyi Moratelli et al., 2013, M. midastactus Moratelli & Wilson, 2014, M. clydejonesi Moratelli et al., 2016, M. attenboroughi Moratelli et al., 2017, M. bakeri Moratelli et al., 2019, and M. armiensis Carrión-Bonilla & Cook, 2020. However, our results indicate that there are at least four haplogroups that might correspond to undescribed species. This scenario confirms the Neotropical region as a highly diverse region for Myotis.

Acknowledgments

We are grateful to G. Garbino, B. Lim, and C. Carrión-Bonilla for the valuable revision of the original text. Support for RLMN and VCC (Ph.D. scholarships) comes from the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil). RM has received support from National Council for Scientific and Technological Development (CNPq, Brazil), and from the Smithsonian Institution (USA). This paper is part of Coordination for the Improvement of Higher Education Personnel the Ph.D. requirements of at the Biodiversity and Evolutionary Biology Graduate Program of the Federal University of Rio de Janeiro.

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Appendix 1

List of specimens examined in the American Museum of Natural History (AMNH, New York, USA); Carnegie Museum of Natural History (CM, Pittsburgh, USA); Field Museum of Natural History (FMNH, Chicago, USA), Louisiana State University, Museum of Zoology (LSUMZ, Baton Rouge, USA); Museu de Zoologia da Universidade de São Paulo (MZUSP, São Paulo, Brazil); Museum of Texas Tech University (TTU, Lubbock, USA); Museum of Vertebrate Zoology, University of California (MVZ, Berkeley, USA); National Museum of Natural History, Smithsonian Institution (USNM, Washington, D.C., USA); Natural History Museum of Los Angeles County (LACM, Los Angeles, USA); Natural History Museum, University of Kansas (KU, Lawrence, USA); and Royal Ontario Museum (ROM, Toronto, Canada). Specimens marked with asterisks were included in the morphometric multivariate analysis.

Myotis albescens (N = 10). Venezuela: Trujillo, Valera, Río Motatán (USNM 370933); Apure, Pto. Páez, Río Cinaruco (USNM 373913); Bolívar, Río Supamo, 50 km SE El Manteco (USNM 387693); Miranda, 7 km E Río Chico, Nr. Pto. Tuy (USNM 387700); Amazonas, Capibara, 106 km SW Esmeralda, Brazo Casiquiare (USNM 409392, 409395); Amazonas, San Juan, 163 km ESE Pto. Ayacucho, Río Manapiare (USNM 409403, 409408, 409410, 409411).

Myotis attenboroughi (N = 13). Trinidad and Tobago: Tobago Island, Charlottesville, 1 km N of Pirate’s Bay, Saint John Parish (USNM 540692 [paratype], 540693 [holotype]); Tobago Island, St. Mary Parish, Hillsborough Reservoir (USNM 538064, 538065, 538066, 538067, 538068, 538069, 540619, 540620, 540621, 540694, 540695 [paratypes]).

Myotis caucensis (N = 22): Colombia: Valle del Cauca, Cauca river (AMNH 32787 [holotype]); Valle del Cauca, Candelaria, Ingenio Mayangüez (USNM 461858–461867). Peru: Cuzco, Madre de Dios, 15 km E Puerto Maldonado, Reserva Cuzco Amazónico (KU 144288–144291); Loreto, Yarinacocha (LSUMZ 12252, 12254–12258).

Myotis clydejonesi (N = 1): Suriname: Sipaliwini, Raleigh Falls (TTU 109227 [holotype]).

Myotis diminutus (N = 2): Ecuador: Los Ríos, Santo Domingo, 47 Km S (By Road), Río Palenque Science Center (USNM 528569 [holotype]). Colombia: Nariño, La Guayacana (LACM 18761).

Myotis handleyi (N = 27). Venezuela: Araguá, Rancho Grande Biological Station, 13 km NW Maracay (USNM 517503, 562923, 562924, 562925, 562926–562933, 562934, 562935, 562936, 562937); Distrito Federal, Pico Ávila, 5 km NE Caracas, near Hotel Humboldt (USNM 370932 [holotype]); Distrito Federal, Pico Ávila, 5 km NE Caracas, near Hotel Humboldt (USNM 370891 [paratype]); Miranda, Curupao, 5 km NW Guarenas (USNM 387723); Monagas, 3 km NW Caripe, near San Agustín (USNM 409391, 409429–409431, 409433, 409435, 409437, 409438).

Myotis keaysi (N = 45). Venezuela: Araguá, Rancho Grande Biological Station, 13 km NW Maracay (USNM 370893–370895, 370898–370902, 370911–370913, 370915– 370922, 370924, 370926, 370929); Araguá, Rancho Grande Biological Station, 13 km NW Maracay (USNM 370927, 370928, 370930, 370931); Araguá, Pico Guayamayo, 13 km NW Maracay (USNM 521564); Araguá, Rancho Grande, Portachuelo (USNM 562920, 563005, 563006); Araguá, Rancho Grande (USNM 562921); Bolívar, Gran Sabana (USNM 130625, 130626); Carabobo, Montalban, 4 km NW Montalban, La Copa (USNM 441741, 441742); Distrito Federal, Los Venados, 4 km NW Caracas (USNM 370889); Distrito Federal, Pico Ávila, 5 km NNE Caracas, near Hotel Humboldt (USNM 370890); Distrito Federal, junction Puerto Cruz Highwayand Colonia Tovar Highway, 0.5 km W (USNM 562984); Guárico, Hacienda El Vira, 10 km NE Altagracia (USNM 387707); Miranda, San Andrés, 16 km SE Caracas (USNM 373920); Miranda, Curupao, 5 km NW Guarenas (USNM 387714–387716, 387718); Monagas, Caripe (USNM 534265).

Myotis larensis (N = 16). Venezuela: Lara, Río Tucuyo (AMNH 130709* [holotype]); Falcón, Capatárida, 6 km SSW (USNM 441710*, 441711*, 441728*, 441735*, 441736*, 441737*, 441740); Zulia, Nr. Cojoro, 35 km NNE Paraguaipoa (USNM 441721*). Guárico (TTU 48162, 48163, 48164, 48168, 48169, 48170); Barinas (CM 78645).

Myotis nesopolus (N = 26). Curaçao: Punda (USNM 101849 [holotype]); Willemstad, Scharloo (USNM 102158); Westpunt, 2.8 km S, 4.5 km E of (CM 52432, 5433*). Bonaire, 8.5 km N, 2 km Wkralendijk (CM 52203, 52204, 52205, 52206, 52207, 52208, 52209, 52211, 52212*, 52213, 52214, 52215, 52216*, 52217*, 52218*, 52219*, 52220*, 52221, 52222*, 52223*, 52224, 52225).

Myotis cf. nigricans (N = 23). Suriname: Para, Zanderij (CM 63933, 69053, 77699). Venezuela: Carabobo, Urama, 10 Km NW Urama, El Central (USNM 140447, 373921–373924, 373926, 373929, 373932, 373933, 373935, 373936, 373942, 373943, 373946, 373947, 373948, 373949, 373950, 441741, 441742).

Myotis oxyotus (N = 9). Venezuela: Amazonas, Cerro Duida, Cano Culebra, 50 km NW Esmeralda (USNM 405799); Amazonas, Cerro Neblina, Camp VII (USNM 560809–560811); Bolívar, Km. 125, 85 km SE El Dorado (USNM387712); Bolívar, El Pauji, 21 km NE Icabaru, El Pauji (USNM441750); Distrito Federal, Alto Ño León, 33 km SW Caracas (USNM 409427); Mérida, La Mucuy, 4 km E Tabay (USNM373919, 387705).

Myotis pilosatibialis (N = 11). Trinidad and Tobago: Trinidad Island, St. George (TTU 5441). Honduras: Francisco Morazán, 1 km W Talanga (LACM 36879 [holotype]). Guatemala: Chimaltenago, Chocoyos (FMNH 41653, 41839, 41840, 41841, 41843, 41844, 41845, 41846, 73365).

Myotis riparius (N = 33). Costa Rica: Puntarenas, 5.3 km S (byroad) San Vito (CM 92491); Limon, Fila La Maquina (LSUMZ 12928). French Guiana: Paracou, near Sinnamary (AMNH 266376, 268591). Guyana: Barima-Waini, North West District (USNM 568021); Potaro-Siparuni, Iwokrama Field Station, Iwokrama Forest (ROM 112049); Potaro-Siparuni, Iwokrama Reserve, Burro Burro River, 25 km WNW of Kurupukari (ROM 107278, 114620); Potaro-Siparuni, Mount Ayanganna, First Plateau Camp (ROM 114688, 114689); Upper Takutu-Upper Essequibo, Gunn’sStrip (ROM 106773). Nicaragua: Chontales (KU 11228). Panamá: Darién, Tacarcuna Village Camp, Río Pucro (USNM 310255 [holotype], 310254, 310256, 310257 [paratypes]); Darién, Rio Paya, Mouth (USNM 306798); Panamá, Cerro Azul (USNM 306795); Chiriquí (USNM 331916); Bocas del Toro, Isla Popa, 1 Km SE Deer Island Channel (USNM 464368). Trinidad and Tobago: Trinidad Island,St. George (TTU 5467). Venezuela: Amazonas,Boca Mavaca, 84 km SSE Esmeralda, 7 km up Río Mavaca (USNM 405803, 405804); Amazonas, Capibara, 106 km SW Esmeralda, Brazo Casiquiare (USNM 409457); Amazonas, ca 2 km SE Cerro Neblina Base Camp (USNM 560625); Amazonas, Tamatama, Río Orinoco (USNM 405806); Apure, Nulita, 29 km SW Santo Domingo, Selvas de San Camilo (USNM 416584, 441746, 441748); Araguá, Rancho Grande (USNM 562940); Barinas, 7 km NE Altamira (USNM 441743); Bolívar, Río Supamo, 50 km SE El Manteco (USNM 387721); Bolívar, San Ignacio de Yhuruani (USNM 448544).

Myotis simus (N = 56). Brazil: Amazonas, Borba (AMNH 91886–91892, 94224, 94225, 94227, 94230–94234); Amazonas, Itacoatiara (MZUSP 4372); Amazonas, Manaus (AMNH 79534, 91472–91478, 91500); Amazonas, Parintins (AMNH 92983, 93489–93497, 93922–93925); Amazonas, Rio Juruá (MZUSP 638, 1074).

Appendix 2

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Specimens used in cytochrome-b analyses, including terminal taxa (focal and putative species of Myotis), GenBank accession numbers of sequences, voucher specimens, localities of origin, and source of information. The information presented for terminal taxonomic identifications results from re-identification of specimens (see Materials and methods), and does not necessarily match those identifications assigned by researchers that generated the corresponding sequence(s) available at GenBank. Abbreviations and acronyms for institutional collections are as follows: American Museum of Natural History, New York, USA (AMNH), Carnegie Museum of Natural History, Pittsburg, USA (CM), Field Museum of Natural History, Chicago, USA (FMNH), Museum of Natural History, University of Kansas, Lawrence, USA (KU), Natural History Museum of Los Angeles County, Los Angeles, USA (LACM), Louisiana State University, Museum of Zoology, Baton Rouge, USA (LSUMZ), Museum of Vertebrate Zoology, University of California, Berkeley, USA (MVZ), University of Nebraska State Museum, Lincoln, USA (UNSM), Muséum national d’Histoire naturelle, Paris, France (MNHN), Národní Muzeum, Prague, Czech (NMP), Museo de Zoología de la Pontificia Universidad Católica del Ecuador, Quito, Ecuador (QCAZ), Royal Ontario Museum, Toronto, Canada (ROM), Universidad Autónoma Metropolitana, Iztapalapa, Mexico (UAMI), Universidade Federal Rural do Rio de Janeiro, Seropédica, Brazil (ALP); and Smithsonian National Museum of Natural History, Washington, DC, USA (USNM). Localities are arranged alphabetically by species and major political unities.

Terminal GenBank Voucher Locality Source
M. albescens JX130444 CM 63920 Nickerie, Suriname Larsen et al. (2012a)
JX130463 TTU 85088 Pastaza, Ecuador Larsen et al. (2012a)
JX130464 TTU 85089 Pastaza, Ecuador Larsen et al. (2012a)
JX130465 TTU 85094 Pastaza, Ecuador Larsen et al. (2012a)
JX130522 TTU 85091 Pastaza, Ecuador Larsen et al. (2012a)
JX130472 TTU 102363 El Oro, Ecuador Larsen et al. (2012a)
JX130500 TTU 102348 El Oro, Ecuador Larsen et al. (2012a)
JX130501 TTU 103744 Guayas, Ecuador Larsen et al. (2012a)
JX130445 TTU 46343 Huánuco, Peru Larsen et al. (2012a)
AF376839 FMNH 162543 Tarija, Bolivia Ruedi and Mayer (2001)
JX130503 TTU 99124 Boquerón, Paraguay Larsen et al. (2012a)
JX130502 TTU 99801 Ñeembucú, Paraguay Larsen et al. (2012a)
JX130504 TTU 99818 Ñeembucú, Paraguay Larsen et al. (2012a)
M. atacamensis AM261882 MVZ 168933 Olmos, Peru Stadelmann et al. (2007)
M. attenboroughi JN020573 UNSM ZM–29470 St. George Parish, Tobago Larsen et al. (2012b)
JN020574 UNSM ZM–29483 St. George Parish, Tobago Larsen et al. (2012b)
M. chiloensis AM261888 – Santiago, Chile Stadelmann et al. (2007)
M. clydejonesi JX130520 TTU 109227 Sipaliwini, Suriname Larsen et al. (2012a)
M. dinellii JX130475 TTU 66489 Córdoba, Argentina Larsen et al. (2012a)
M. dominicensis AF376848 – St. Joseph’s Parish, Dominica Ruedi and Mayer (2001)
JN020554 TTU 31519 St. Joseph’s Parish, Dominica Larsen et al. (2012b)
JN020555 TTU 31507 St. Joseph’s Parish, Dominica Larsen et al. (2012b)
JN020556 TTU 31508 St. Joseph’s Parish, Dominica Larsen et al. (2012b)
M. larensis JN020569 TTU 48161 Guárico, Venezuela Larsen et al. (2012b)
JX130529 TTU 48162 Guárico, Venezuela Larsen et al. (2012a)
JX130530 – Guárico, Venezuela Larsen et al. (2012a)
JX130531 TTU 48163 Guárico, Venezuela Larsen et al. (2012a)
JX130532 TTU 48164 Guárico, Venezuela Larsen et al. (2012a)
JX130533 TTU 48168 Guárico, Venezuela Larsen et al. (2012a)
JX130535 CM 78645 Guárico, Venezuela Larsen et al. (2012a)
JX130543 TTU 48169 Guárico, Venezuela Larsen et al. (2012a)
JX130543 TTU 48169 Guárico, Venezuela Larsen et al. (2012a)
M. lavali AF376864 MVZ AD50 Paraíba, Brazil Ruedi and Mayer (2001)
M. levis AF376853 FMNH 141600 São Paulo, Brazil Ruedi and Mayer (2001)
M. martiniquensis AM262332 – Martinique Stadelmann et al. (2007)
JN020558 MNHN:2005–896 Le Morne–Rouge, Martinique Larsen et al. (2012b)
M. martiniquensis JN020557 MNHN:2005–895 GranďRivière, Martinique Larsen et al. (2012b)
JN020559 – GranďRivière, Martinique Larsen et al. (2012b)
JN020560 MNHN:2008–974 GranďRivière, Martinique Larsen et al. (2012b)
JN020561 – GranďRivière, Martinique Larsen et al. (2012b)
M. nesopolus JN020575 – Bonaire, Netherlands Antilles Larsen et al. (2012b)
JN020576 – Bonaire, Netherlands Antilles Larsen et al. (2012b)
JN020577 – Bonaire, Netherlands Antilles Larsen et al. (2012b)
M. nigricans JX130450 TTU 34952 La Paz, Bolivia Larsen et al. (2012a)
JX130528 TTU 34953 La Paz, Bolivia Larsen et al. (2012a)
JX130455 TTU 95992 San Pedro, Paraguay Larsen et al. (2012a)
JX130496 TTU 99743 Presidente Hayes, Paraguay Larsen et al. (2012a)
JX130498 TTU 99046 Alto Paraguai, Paraguay Larsen et al. (2012a)
JX130499 TTU 99802 Ñeembucú, Paraguay Larsen et al. (2012a)
JX130539 TTU 99516 Concepciόn, Paraguay Larsen et al. (2012a)
JX130540 TTU 99151 Boquerón, Paraguay Larsen et al. (2012a)
M. nyctor JN020562 CM 83427 St. David Parish, Grenada Larsen et al. (2012b)
JN020563 TTU 109225 St. Thomas Parish, Barbados Larsen et al. (2012b)
JN020564 TTU 109226 St. Thomas Parish, Barbados Larsen et al. (2012b)
JN020565 TTU 109229 St. Thomas Parish, Barbados Larsen et al. (2012b)
JN020566 TTU 109224 St. Thomas Parish, Barbados Larsen et al. (2012b)
JN020567 TTU 109230 St. Thomas Parish, Barbados Larsen et al. (2012b)
M. oxyotus AF376865 FMNH 129208 Lima, Peru Ruedi and Mayer (2001)
M. pilosatibialis JX130449 TTU 47514 Yucatán, Mexico Larsen et al. (2012a)
JX130525 – Yucatán, Mexico Larsen et al. (2012a)
AF376852 – Yucatán, Mexico Ruedi and Mayer (2001)
JX130489 CM 55764 Vera Cruz, Mexico Larsen et al. (2012a)
M. elegans JX130479 TTU 84380 Atlántida, Honduras Larsen et al. (2012a)
JX130480 TTU 84138 Atlántida, Honduras Larsen et al. (2012a)
M. riparius AM261891 – La Selva, Costa Rica Stadelmann et al. (2007)
JX130474 CM 78659 Bolívar, Venezuela Larsen et al. (2012a)
JX130473 CM 68443 Para, Suriname Larsen et al. (2012a)
JX130469 TTU 85344 Esmeraldas, Ecuador Larsen et al. (2012a)
JX130515 TTU 85345 Esmeraldas, Ecuador Larsen et al. (2012a)
JX130572 TTU 102681 Esmeraldas, Ecuador Larsen et al. (2012a)
JX130492 TTU 102883 Esmeraldas, Ecuador Larsen et al. (2012a)
JX130513 TTU 84870 Pastaza, Equador Larsen et al. (2012a)
JX130506 TTU 85090 El Oro, Equador Larsen et al. (2012a)
JX130516 QCAZ 11380 Chimborazo, Equador Larsen et al. (2012a)
JX130436 – Huánuco, Peru Larsen et al. (2012a)
JX130481 TTU 46348 Huánuco, Peru Larsen et al. (2012a)
AF376866 MVZ AD119* Pernambuco, Brazil Ruedi and Mayer (2001)
AF376867 MVZ AD472* São Paulo, Brazil Ruedi and Mayer (2001)
AM262336 – São Paulo, Brazil Stadelmann et al. (2007)
JX130485 TTU 99645 Paraguari, Paraguay Larsen et al. (2012a)
JX130486 TTU 94912 Canindeyu, Paraguay Larsen et al. (2012a)
M. riparius JX130488 TTU 122454 Canindeyu, Paraguay Larsen et al. (2012a)
JX130491 TTU 99378 Canindeyu, Paraguay Larsen et al. (2012a)
M. velifer EF222340 TTU 48587 Texas, USA Baird et al. (2008)
EU680299 TTU 44818 Texas, USA Baird et al. (2008)
JX130468 TTU 109261 Texas, USA Larsen et al. (2012a)
AF376870 MVZ 146766 Sonora, Mexico Ruedi and Mayer (2001)
JX130478 TTU 44816 Tamaulipas, Mexico Larsen et al. (2012a)
JX130438 UAMI 15306 Michoacán, Mexico Larsen et al. (2012a)
JX130462 UAMI 15304 Michoacán, Mexico Larsen et al. (2012a)
JX130589 UAMI 15305 Michoacán, Mexico Larsen et al. (2012a)
JX130592 – Michoacán, Mexico Larsen et al. (2012a)
JX130477 TTU 60983 Santa Ana, El Salvador Larsen et al. (2012a)
M. vivesi AJ504406 – Gulf of California, Mexico Stadelmann et al. (2004)
AJ504407 – Gulf of California, Mexico Stadelmann et al. (2004)
M. yumanensis AF376875 MVZ 15585 California, USA Ruedi and Mayer (2001)
M. sp. 1 JX130523 TTU 103803 El Oro, Ecuador Larsen et al. (2012a)
JX130541 TTU 103751 El Oro, Ecuador Larsen et al. (2012a)
JX130546 TTU 102760 El Oro, Ecuador Larsen et al. (2012a)
JX130547 TTU 102765 El Oro, Ecuador Larsen et al. (2012a)
JX130548 TTU 102487 El Oro, Ecuador Larsen et al. (2012a)
JX130549 TTU 102489 El Oro, Ecuador Larsen et al. (2012a)
JX130550 TTU 102490 El Oro, Ecuador Larsen et al. (2012a)
M. sp. 2 JX130452 TTU 46347 Huánuco, Peru Larsen et al. (2012a)
JX130537 TTU 46344 Huánuco, Peru Larsen et al. (2012a)
JX130538 TTU 46346 Huánuco, Peru Larsen et al. (2012a)
M. sp. 3 JX130493 TTU 61228 Valle, Honduras Larsen et al. (2012a)
M. sp. 4 JN020570 CM 63933 Nickerie, Suriname Larsen et al. (2012b)
JN020571 CM 69053 Para, Suriname Larsen et al. (2012b)
JN020572 CM 77699 Para, Suriname Larsen et al. (2012b)
Outgroups
M. brandtii AF376844 – Neuhaus, Germany Ruedi and Mayer (2001)
AM261886 NMP PB 916 North west, Russia Stadelmann et al. (2007)
AY665139 – Moscow, Russia Tsytsulina et al. (2012)
AY665168 – Znojmo, Czech Republic Tsytsulina et al. (2012)
M. gracilis AB106609 – Hokkaido, Japan Kawai et al. (2003)
AB243025 – Hokkaido, Japan Kawai et al. (2006)
AB243026 – Hokkaido, Japan Kawai et al. (2006)
AB243027 – Hokkaido, Japan Kawai et al. (2006)
AB243028 – Hokkaido, Japan Kawai et al. (2006)
AB243029 – Hokkaido, Japan Kawai et al. (2006)
AB243030 – Hokkaido, Japan Kawai et al. (2006)
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