Taxonomic status assessment of the Mexican populations of funnel-eared bats, genus Natalus...

INTRODUCTION

Due to its morphological conservatism, theAmerican funnel-eared bat genus Natalus has had a complex taxonomic history with an uncertainty inthe number of species, which has ranged from two,N. stramineus Gray, 1838, and N. tumidirostris Mil -ler, 1900 (Koopman, 1994) to eight: N. espiritosan-tensis (Ruschi, 1951), from Brazil, Bolivia and Para -guay; N. jamaicensis (Goodwin, 1959), from Ja mai -ca; N. lanatus Tejedor, 2005, from Mex ico andCosta Rica; N. major Miller, 1902, from the Domi -ni can Republic and Haiti; N. mexicanus Miller,1902, distributed from Mexico to Panama includingseveral offshore continental or oceanic islands ofMex ico, Panama, and Colombia; N. primus Antho -ny, 1919, from Cuba, Isle of Pines, the Bahamas,and Grand Cayman; N. stramineus Gray, 1838, from the islands of the Lesser Antilles north of theSt. Lucia Chanel; and N. tumidirostris Miller, 1900,

found in Colombia, Venezuela, Guyana, Suriname,French Guiana, and the islands of Trinidad, Tobago,Margarita, Curacao, and Bonaire (Tejedor, 2011).

In Mexico, the taxonomic status of the funnel-eared bats of the genus Natalus has also been un-clear, with different researchers proposing contrast-ing taxonomic arrangements. Only one species, N. stramineus, has been generally recognized inMexico with two allopatric subspecies, N. s. stra mi -neus and N. s. mexicanus (Hall, 1981), which weredifferentiated by morphological features such as col-oration and body size, but with recognized incon-sistencies (Dalquest, 1950; Goodwin, 1959; Villa-Ramírez, 1966; Hall, 1981).

The most recent taxonomic arrangement sug-gests the presence of two American funnel-earedbat species in Mexico: N. lanatus and N. mexicanus(Tejedor, 2006). Natalus lanatus, which was de-scribed one year earlier in a taxonomic review basedon museum specimens (Tejedor, 2005), displays

Acta Chiropterologica, 14(2): 305–316, 2012PL ISSN 1508-1109 © Museum and Institute of Zoology PAS

doi: 10.3161/150811012X661639

Taxonomic status assessment of the Mexican populations of funnel-eared bats,

genus Natalus (Chiroptera: Natalidae)

RICARDO LÓPEZ-WILCHIS1, 4, LUIS M. GUEVARA-CHUMACERO1, 3, NEÓFITO ÁNGELES PÉREZ1, JAVIER JUSTE3,CARLOS IBÁÑEZ3, and IRENE D. L. A. BARRIGA-SOSA2

1Departamento de Biología, Universidad Autónoma Metropolitana — Iztapalapa, Av. San Rafael Atlixco N° 186, Col. Vicentina, C.P. 09340, Iztapalapa, México D.F.

2Departamento de Hidrobiología, Universidad Autónoma Metropolitana — Iztapalapa, Av. San Rafael Atlixco N° 186, Col.Vicentina, C.P. 09340, Iztapalapa, México D.F.

3Estación Biológica de Doñana, C.S.I.C., A.P. 1056, 41080 Sevilla, España4Corresponding author: E-mail: [email protected]

Due to its morphological conservatism, the American funnel-eared bats of the genus Natalus have had a complex taxonomic history.In Mexico only one species, N. stramineus, has traditionally been recognized. But recently a new endemic species, N. lanatus, wasdescribed in the region as a sympatric sibling species of N. stramineus. Natalus stramineus, with a geographic distribution extendingfrom Mexico to Panama, was subsequently reassigned to N. mexicanus. In this study, we analyzed morphological and molecularcharacters to determine if two distinct species of funnel-eared bats (N. lanatus and N. mexicanus) occur in Mexico. We found thatthe proposed diagnostic morphological characters that separate the two taxa are not consistent and rarely differentiate between thetwo forms. Likewise, phylogenetic analyses do not support the separation of Natalus into two species in Mexico, but there is clearlygeographic structure when all Mexican haplotypes are combined. The genetic distance between the haplotypes that could be ascribedto N. lanatus and N. mexicanus was typically within the range of conspecifics and not distinct species. To summarize, the results ofboth morphological and molecular analyses point to the presence of a single species of Natalus in Mexico.

Key words: bats, cytochrome b, Mexico, sibling species, taxonomy

diagnostic characters that include bicolored ventralfur; dorsal and ventral hairs are always darker at thebase than at the tips; and dense pelage, woolly, anddull, grayish to ochraceous. According to the taxo-nomic description, the most distinctive features ofthis new species are the presence of conspicuoustufts of relatively long bristles projecting from thebase of each claw and the presence of 0–1 pleats onthe lateral margin of the ear. Natalus lanatus was described as endemic to Mexico, particularly thestates of Chihuahua, Durango, Sinaloa, Nayarit, Ja -lis co, Guerrero and Veracruz; however, recentlyRodrí guez-Herrera et al. (2011) recorded its occur-rence in Costa Rica.

In contrast, N. mexicanus shows monocolor ven-tral pelage and a bicolored dorsal pattern, whenpresent, with individual hairs darker at the tips than at the base; legs sparsely haired, with the tips ofthe toes lacking long hairs and dense tufts; and theear presenting 2–4 pleats on its lateral margin(Tejedor, 2005). This species is reported from Los Cabos, Baja California and two separated nar-row strips that extend along the Pacific coast(Sonora to Chiapas) and the Gulf of Mexico(Tamaulipas to Tabasco); the two strips converge in the lowlands of the Isthmus of Tehuantepec andthen continue southeast into the Yucatan Penin-sula and down to Panama (Tejedor, 2011). Accord -ing to Tejedor (2005, 2011) morphometric skullcharacters cannot be used to separate N. lanatusfrom N. mexicanus.

Arroyo-Cabrales et al. (1997) made the firstanalysis of the genetic variation of Natalus fromMexico, Belize and the Dominican Republic, basedon allozymes. These authors identified the pres-ence of two lineages in Mexico, one in the North and another in the South, and they concluded thatthe differentiation was likely related to the widerange of ecological and environmental conditions in which the species was found. More recent-ly, Dávalos (2005), in a phylogenetic analysis of the Natalidae family, suggested that a detailed geographical and historical analysis of all Mexi-can and Central American lineages is required to re-solve the systematics of the genus Natalus in this region.

The use of molecular tools developed in recentyears has played an important role in establishinglimits between species (Bradley and Baker, 2001;Ruedi and Mayer, 2001), unveiling cryptic biodiver-sity that remained hidden in many groups of plantsand animals when analyzed using traditional meth-ods (Whittall et al., 2004; Cepicka et al., 2005;

Baker and Bradley, 2006). Cryptic diversity hasbeen shown to be particularly common in groupssuch as bats (Ibáñez et al., 2006; Mayer et al., 2007).The mitochondrial DNA gene cytochrome b (cyt b)is one of the most commonly used genes for clarify-ing phylogenetic relationships at the inter-specificlevel for bats (Hoffmann and Baker, 2001; Tian etal., 2004; Dávalos, 2005; Baker and Bradley, 2006;Ibáñez et al., 2006). In this study we present analy-ses of morphological and molecular characters toclarify the taxonomic status of the genus Natalusand to confirm the presence of two species of Amer -ican funnel-eared bats in Mexico.

MATERIALS AND METHODS

Sampling and Taxonomic Assignment

An extensive sampling of funnel-eared bats was conductedacross Mexico, including several of the localities reported byTejedor (2005) in the description of N. lanatus. The bats wereidentified as N. lanatus and N. mexicanus according to the diag-nostic characters proposed by Tejedor (2005), namely ventraland dorsal pelage patterns, the presence of tufts of hair on thebase of the toenails and the number of pleats on the lateral mar-gin of the ear. Various individuals presented a mixture of mor-phological characteristics of both species and were thereforedifficult to assign to either of the two proposed taxa. These in-dividuals were recorded as ‘ambiguous’. All bats were sexed,measured and weighed.

Mexican funnel-eared bats were collected throughout thedistribution range of both species (Table 1 and Fig. 1). Wing-punches from captured bats were obtained using a 3-mm biop-sy punch (Fray Products Corp., Buffalo, New York, USA), andpreserved in 70% ethanol. A few biopsied specimens were keptas vouchers and deposited in the collections at the EstaciónBiológica de Doñana (CSIC) and Universidad AutónomaMetropolitana — Iztapalapa (catalogue numbers: EBD28506–EBD28513 and RLW1994, RLW1996-2001, respectively).

Natalus lanatus and N. mexicanus are considered relativelyabundant and not endangered (Secretaría de Medio Ambiente yRecursos Naturales, 2002), and are listed as ‘Least Concern’(Arroyo-Cabrales and Alvarez, 2008; Velazco and Pineda,2008). All appropriate ethics and other approvals were obtainedfor the research.

Morphological Analyses

We used 23 individuals of N. stramineus and 10 N. lanatusfor the morphological analyses. Forearm (FA) length was meas-ured with a Mitutoyo vernier caliper (0.01 mm). Differences inFA between adults of N. lanatus and N. mexicanus were exam-ined by a 2-way ANOVA design, considering FA and Sex asfixed factors. The homogeneity of variance was confirmed us-ing Levene’s test (results not shown). All statistics were per-formed with Stata 11.0 software (Stata Corp., College Station,Texas, USA). Finally, a few voucher specimens were inspectedunder a Zeiss stereo microscope (10×/23 mm) and photo -graphed with AxioCam ICc3 (3.3 megapixel resolution).

306 R. López-Wilchis, L. M. Guevara-Chumacero, N. Ángeles Pérez, J. Juste, C. Ibáñez, et al.

DNA Extraction and Sequencing

Prior to DNA extraction, the tissues were washed with ster-ile water. Total DNA was then extracted using the PromegaWizard Genomic DNA purification Kit, according to the manu-facturer’s instructions (Promega Corp., Madison, Wisconsin,USA). The final product was diluted in 70 µl of sterile water andstored at -20°C for further analyses. We amplified the first threedomains of the cyt b gene via polymerase chain reaction (PCR− Saiki et al., 1998), using the primer pairs MVZ05 (Smith and Patton, 1993) and NEW12 (Dávalos and Jansa, 2004).Reactions were carried out in a total volume of 25 μl with approximately 20–40 ng of template DNA, 2 μl of 10 × buffer,

10 mM of each primer, 200 mM of each dNTP, 2 mM of MgCl2,and 2.5 units of Taq DNA polymerase (MBI Fermentas). PCRcycles were as follows: 3 min denaturation at 95°C; 35 cycles ofdenaturation at 95°C for 20 s, annealing at 50.5°C for 15 s, andextension at 72°C for 1 min; final extension of 7 min at 72°C.Sequences were aligned using BioEdit (Hall, 1999), with subse-quent visual confirmation.

Phylogenetic Analyses

Five hundred base pairs (bp) of the cyt b gene were se-quenced from each individual (n = 52). Phylogenetic recon-structions were obtained under Maximum Parsimony (MP) and

Taxonomic assessment of Natalus 307

TABLE 1. Details of the studied populations of Mexican funnel-eared bats of the genus Natalus. Locality name with acronym in parentheses; Mexican state; geographical coordinates; number of bats sampled

Locality State Latitude Longitude nPescadero (PE) Baja California Sur 23º20.72’N 110º07.56’W 6Santiago (SA) Baja California Sur 23º32.21’N 109º40.35’W 4Rosario (RO) Sinaloa 23º04.32’N 105º58.03’W 3Etzatlán (ET) Jalisco 21º02.24’N 104º21.52’W 4Juxtlahuaca (JU) Guerrero 17º25.43’N 99º08.51’W 5Cardona (CA) Colima 19º21.66’N 103º65.00’W 8Tuxtla Gutiérrez (TG) Chiapas 16°49.32’N 93°09.12’W 5San Sebastián (SS) Oaxaca 16º63.00’N 96°96.90’W 5Troncones (TR) Tamaulipas 23º46.20’N 99º12.17’W 2Jiménez (JI) Tamaulipas 25º50.39’N 97º31.41’W 3Cántaro (CAN) Veracruz 19°13.78’N 96°38.24’W 7

FIG. 1. Location of the sampled populations of Natalus in Mexico (acronyms in Table 1). Squares indicate the localities followingthe taxonomic categorization of the different populations according to the diagnostic characters proposed by Tejedor (2005) (white — N. lanatus; black — N. mexicanus; striped — ‘ambiguous’). The distribution area for Natalus in Mexico is shown in gray

110°W 100°W 90°W

30°N

20°N

N. lanatus

N. mexicanus

ambiguous

Maximum Likelihood (ML) criteria with PAUP* 4.0b10(Swofford, 2002), and with a Bayesian approach in MrBayes3.0 (Huelsenbeck and Ronquist, 2001). In accordance withDávalos (2005), trees were rooted using sequences from Myotis velifer (AF376870) and Chilonatalus micropus(AY621026), obtained from GenBank. We also included se-quences obtained from GenBank of the sister species, Natalusmajor (AY621021), N. jamaicensis (AY621023), and N. tumidi -rostris (AY621009).

Under MP, trees were obtained considering gaps as a fifthstate and after heuristic search with an initial tree obtained viastepwise addition (random input order) of the taxa, followed bya complete tree-bisection–reconnection (TBR) branch swap-ping. The robustness of the topology was then assessed throughbootstrapping (Felsenstein, 1985) after 1,000 replicates. Themost appropriate model of nucleotide evolution was selected us-ing ModelTest 3.7 (Posada and Crandall, 1998). A ML heuristicsearch was then conducted using this model. Again, the robust-ness of the topology was evaluated using bootstrap resampling(Felsenstein, 1985) after 1,000 replicates. Both MP and MLanalyses were performed in PAUP* 4.0b10 (Swofford, 2002).Bayesian inference was conducted with random starting treeswithout constraints and specifying the selected substitutionmodel, and allowing specific parameters to be estimated. TheBayesian topologies were obtained after five simultaneousMarkov chains were run for two million generations; trees weresampled every 100 generations. The resulting burn-in valueswere determined empirically after tree likelihood scores reachedstationary. Analyses were repeated in two separate runs to en-sure that trees converged on the same topology and similar pa-rameters. A 50% majority rule consensus tree was then obtainedin which each clade’s posterior probability value is indicative ofthe proportion of samples that recover that particular clade(Huelsenbeck and Ronquist, 2001).

We estimated Kimura’s 2-parameter (K2P) genetic dis-tances between the two proposed species and in relation to the‘ambiguous’ group using the program MEGA 4.0 (Tamura etal., 2007). Different studies have used this particular model tomeasure genetic distances in bats and establish relative levels ofdifferentiation at the inter-specific level (Bradley and Baker,2001; Lewis-Oritt et al., 2001; Baker and Bradley, 2006; Ibáñezet al., 2006; García-Mudarra et al., 2009).

Finally, we assessed the existence of hierarchical geneticstructure in Natalus populations by an analysis of molecularvariance (AMOVA — Excoffier et al., 1992) with genetic di ver -gence values (FST). AMOVA and a bootstrap analysis with 5,000replicates were performed in Arlequin (Schneider et al., 2000).

RESULTS

Morphological Analyses

A total of 52 samples of Natalus bats were ana-lyzed for this study, representing 11 populations dis-tributed throughout the whole range of the genus in Mexico (Fig. 1). According to the diagnostic characters defined by Tejedor (2005, 2011), only 10 (19.2%) bats could be identified unambiguouslyas N. lanatus and 29 (55.8%) as N. mexicanus; whereas 13 (25.0%) bats showed a mixture, withouta defined pattern, of external attributes of both

spe cies and were included within an ‘ambiguous’third group. Only two populations, Rosario and SanSeba stián, were monotypic and comprised exclu-sively of individuals with characteristics of N. lana-tus; while four populations, namely Juxtlahuaca,Tuxtla Gu tiér rez, Troncones and Jiménez, presentedsolely N. me xicanus characteristics. In contrast, onlythe Car do na population, consisted of both N. lanatusand N. me xi canus individuals, and the four remain-ing populations comprised ‘ambiguous’ bats with a combination or mixture of characters from bothproposed taxa (Table 2). According to the two-wayANOVA, no differences in adult FA length werefound between N. lanatus (4 YY, 6 XX) and N. me -xicanus (13 YY, 10 XX— F = 0.18, P = 0.67), be-tween sexes (F = 0.01, P = 0.92) or their interaction(F = 0.04, P = 0.84; in all cases d.f. = 1).

Molecular Analyses

The sequence alignment showed 27 variable sitesand 18 parsimony informative sites. Twenty-threedistinct haplotypes were identified, 52.2% of themwere shared, whereas 47.8% were unique haplo-types. The most common haplotypes were H2 andH7, found in 15 individuals. Haplotype 2 was foundin three localities (Table 2 and Appendix).

A TrN model (Tamura and Nei, 1993) with gam-ma distribution (shape parameter = 0.1567) waschosen for our cyt b sequences. ML, MP and theBayesian reconstructions were very similar in their topologies and showed: 1) monophyly for all Mexican Natalus, 2) random allocation of haplotypes between the putative species lineagesbased on the morphological characters previouslysuggested, and 3) geographic structure (Fig. 2). Cor -rected genetic distance between haplotypes identi-fied as N. lanatus and N. mexicanus was 1.18%;whereas among distinct Natalus species this valueranged from 3.67% to 5.45% (Table 3). Within theMexican natalids the average corrected distance was1.14% for N. lanatus, 0.75% for N. mexicanus, and1.08% for ‘ambiguous’ specimens. Significant sepa-ration between populations was also supported byAMOVA, with 79.4% of the variance found amongpopulations (FST = 0.794, P < 0.05), and only 20.6%found within populations.

DISCUSSION

When applying the proposed diagnostic charac-ters (Tejedor, 2005) to our sample of bats we foundthat two populations were entirely composed of



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Na talus lanatus individuals, four populations con-sisted exclusively of N. mexicanus individuals, oneshowed the presence of both taxa, and four popula-tions comprised specimens with a mixed combina-tion of diagnostic characters (Fig. 3). Pelage color

was an effective diagnostic character for very fewbats due to the high level of inter- and intra-specificvariation (Fig. 3A1–A7 and B1–B7). The use of finedetails of pelage coloration demands the examina-tion of a large series of specimens (Borisen ko et al.,


Taxon N. jamaicensis N. major N. tumidirostris N. lanatus N. mexicanusN. jamaicensis − − − − −N. major 4.80 − − − −N. tumidirostris 5.45 5.24 − − −N. lanatus 4.94 4.49 3.67 − −N. mexicanus 4.80 4.42 3.90 1.18 −

TABLE 3. Corrected genetic distances (K2P) in the cyt b between the groups of bats ascribed to the species of N. lanatus and N. mexicanus according to the diagnostic characters following Tejedor (2005)

FIG. 3. Variation observed in diagnostic characters among seven selected specimens from the population of ‘El Cántaro’, Veracruz.Dorsal pelage, varying from monocolor to tricolor, and presenting different coloration stages ranging from buffish and yellowish todeep chestnut or dark red (A1–A7). Ventral pelage for the same set of specimens is monocolor in all individuals (B1–B7). Variationof diagnostic characters in one individual in chestnut phase (A7, B7): dorsal pelage is tricolor as described for N. lanatus (C1);ventral pelage is monocolor as reported for N. mexicanus (C2); the medial margin of the ear is straight as observed in N. lanatus, butwith three pleats (see arrow) as described for N. mexicanus (C3); and claws with long tufts of hairs as reported for N. lanatus (C4). D1–D4: Variation of diagnostic characters in an individual in red phase (A1–B1): both dorsal and ventral pelages are monocolor asreported for N. mexicanus (D1–D2); medial margin of the ear is concave (D3) as described for N. mexicanus; and claws with long

tufts of hairs as observed in N. lanatus (D4)

A1 A2 A3 A4 A5 A6 A7

B1 B2 B3 B4 B5 B6 B7

C1 C2 C3 C4

D1 D2 D3 D4

tricolor monocolor 3 pleats tufts

monocolor monocolor ear concave tufts

2008), and it is well known that this character is difficult to assess qualitatively given the high degreeof inevitable subjectivity associated with the re-searchers’ perception of color (Caro, 2005; Davisand Castleberry, 2010). In this study we found allpossible combinations between diagnostic charac-ters (Fig. 3), such as individuals presenting ventralpelage monocolor as observed in N. mexicanusbut tricolor dorsal pelage as described for N. la-natus (Fig. 3C1–C2), or individuals displaying dor-sal and ventral pelage monocolor as reported for N. me xicanus (Fig. 3D1–D2) but combined with hairtufts at the base of the claws as described for N. la -natus (Fig. 3D4). On the one hand, it is important toconsider that Natalus species generally roost in largenumbers, and prefer to occupy warm and humidparts of the roost (Torres-Flores and Ló pez-Wilchis,2010), conditions that promote high con centrationsof ammonia gas and water vapor, which could causevariations in pelage color, as suggested by Con stan -tine (1957, 1958). In addition, it is important to takeinto account the possible effect of time on the col-oration of museum specimens (Davis and Castle -berry, 2010) keeping in mind that most of the speci-mens of Natalus from Mexico examined by Tejedor(2005) have been stored in the scientific collectionsof the AMNH for over 35 years (López-Wilchis,2003) and even the proposed holotype of N. lanatuswas collected in 1950, more than 60 years ago.

A high degree of subjectivity is also unavoidablein relation to the shape of the ear margin and thenumber of pleats. In this study, some specimens witha fur coloration pattern characteristic of N. lanatusshowed the concave medial margin of the ear (Fig.3D3) and three ear pleats (Fig. 3C3), typically re-ported for N. mexicanus. Similarly, as much as 25%of the Natalus specimens from Santiago’s popula-tion, which are categorized as N. mexicanus accord-ing to the bristle of the toes, presented the straightmedial margin, broader apex, and lack of ear pleatscharacteristic of N. lanatus. Tejedor (2011) empha-sizes that the only distinct external diagnostic character of N. mexicanus is the shape of the medialmargin of the ear; however, great care must be tak-en in analyses utilizing museum skins because thenetwork of bundles of collagen and elastic fibersthat maintains ear shape may deform and denaturewith exposure to formalin or alcohol (Holbrook andOdland, 1978; Myers, 1978; Viidik, 1980). More -over, in live specimens the pinna changes regularlybetween upright and bent configurations, and thusthe curvature of large portions of the ear surface alsochanges (Gao et al., 2011). In our experience, the

easiest character to record in the field was the pres-ence or absence of hair tufts on the base of the toes.Nevertheless, this feature was not consistent withthe remaining characters in distinguishing the proposed taxa. To summarize, our results show a high degree of ambiguity for all diagnostic mor-phological characters, making them unreliable forthe proposed distinction between the two species.

Regarding size, no differences were found be-tween N. lanatus and N. mexicanus with respect toFA length, even after taking into account possiblesex-based differences. These results lead us to ques-tion the utility of such diagnostic characters or eventhe validity of the hypothesis of two Natalus speciesin Mexico.

None of the phylogenetic reconstructions sup-ported the separation of two lineages as would beexpected if the samples belonged to two actual spe -cies; on the contrary, the haplotypes showed a ran-dom distribution without association with morpho-logical species suggested by Tejedor (2005; see Fig.2). The resulting cyt b tree topology does not strong-ly support a single lineage; rather it looks as if thereare three clades. What causes this topology is notpresently clear. It could be one species with geo-graphic variation or one lineage with rare mutations,two species with poorly expressed characters, orthree taxa yet to be defined.

The first proposition is supported by AMOVA(FST = 0.794, P < 0.05) with a geographic structureas show in Fig. 2 (right side). The presence of a sin-gle lineage, with rare mutations, is reinforced by theconclusion obtained of Clare et al. (2011), using a different mitochondrial gene (COI), for Natalusspecimens from Southeastern Mexico, Guatemalaand Nicaragua. Nevertheless, results on the geo-graphic structure or the presence of rare mutations insome populations need to be confirmed through a more detailed study.

The possibility of two species is justified only ifboth the presence of interconnected or shared haplo-types between N. lanatus and N. mexicanus (H6 andH2, respectively) is due to an incomplete lineagesorting and the ‘ambiguous’ individuals are consid-ered hybrids. However, this hypothesis is difficult toaccept, given that this is a not migratory species andthere is more than an 800 km distance between hap-lotypes H14–H17 (Central Valley of Oaxaca) andH2 and H6 (Pacific slope), belonging to N. lanatus.In order to confirm the presence of hybrids it is nec-essary to use a fast evolving marker as micro-satellites or AFLP (e.g., Berthier et al., 2006; Larsenet al. 2010; Clare, 2011). The hypothesis of three


lineages is not supported by the low genetic differ-entiation between them (K2P distance range variedbetween 1.29% and 1.66%).

Although genetic distances provide just an in-dicative taxonomic value and cannot be used tomake direct taxonomic inferences (Jones et al.,2005), in our study the cyt b corrected genetic dis-tances found among species of Natalus are similar to the lowest values reported by Baker and Bradley(2006) for sister species of bats (from 3.3% for My -otis to 12.8% for Vampyriscus) and are lower thanthat reported by Dávalos (2005) specifically forNata lus (ca. 5.0%). The observed distance betweenthe putative species N. lanatus and N. mexicanus(1.18%) was clearly below the minimum value re-ported by Baker and Bradley (2006) for relatedspecies of bats; in fact, this distance value is moresimilar to the average value reported for withinspecies variation. The genetic distance between bothspecies is consistent with a single gene-pool sce-nario. Clare et al. (2011), using a different mito-chondrial gene (COI), proposed a single lineage forNatalus in Mexico and Central America.

As can be seen, this study has failed to recoverthe distinct molecular lineages or species that wouldbe expected according to the previously proposedspecies differentiation within the Mexican Natalus.A comparison of the recovered patterns of geneticvariation shows that the mitochondrial lineages ex-hibit no discernible pattern in habitat preferences. It is interesting to note that the specimens used inthis study and assigned to N. lanatus, N. mexicanusor an ‘ambiguous’ group were found in all sorts ofenvironments along a wide altitudinal range (fromsea level to 1,700 m), varying from pine-oak foreststo deciduous forests to arid tropical scrubs. Theseobservations are similar to the results obtained byTorres-Flores and López-Wilchis (2010) and sup-port the high environmental plasticity of the speciesalready noted by Arroyo-Cabrales et al. (1997).

In conclusion, the results of both the morpholog-ical and genetic analyses clearly demonstrate thatthe proposed taxonomic distinction of N. lanatusand N. mexicanus is not sustained, and instead, allMexican Natalus correspond to a single taxon. As a consequence, all Mexican Natalus should be re-ferred to as N. mexicanus according to the last revi-sion of the genus (Tejedor, 2006, 2011).

ACKNOWLEDGEMENTS

We would like to thank R.M. Aguilar, W. Torres Flores, L.A. Martínez Méndez, D. G. Zárate Martínez, I. Pérez Espinosa, M. A. León Galván and the many local people who assisted

during the fieldwork. J. M. García-Mudarra helped in the labo-ratory. I. Hazarmabeth Salgado Ugarte provided advice on theexplor atory analysis of statistical data. We thank anonymous reviewers for their helpful comments and suggestions. Thiswork was supported by the División de Ciencias Biológicas y dela Salud, Universidad Autónoma Metropolitana and ConsejoNacional de Ciencia y Tecnología (CONACYT) fellowship forLMG-C (126899, 150712), and support for RL-W (CB-2009-01/128459).

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Received 01 August 2012, accepted 22 October 2012

APPENDIX

List of taxa, localities, countries, haplotypes and GenBank accession numbers of cyt b (voucher number), and references of thesamples used for the study. For the names of localities in Mexico see Table 1

Species Location Country Cyt b haplotypes / Genbank(voucher) Reference

Natalus mexicanus JU Mexico H1 / H1285420 Present studyN. mexicanus JU Mexico H2 / HQ285421 Present studyN. mexicanus JU Mexico H2 / HQ285421 Present studyN. mexicanus JU Mexico H2 / HQ285421 Present studyN. mexicanus JU Mexico H3 / HQ285422 Present studyN. mexicanus ET Mexico H2 / HQ285421 Present studyN. mexicanus ET Mexico H4 / HQ285423 Present studyN. mexicanus ET Mexico H4 / HQ285423 Present studyN. mexicanus ET Mexico H5 / HQ285424 Present studyN. mexicanus RO Mexico H6 / HQ285425 (EBD28512) Present studyN. mexicanus RO Mexico H6 / HQ285425 (EBD28513) Present studyN. mexicanus RO Mexico H6 / HQ285425 (EBD28511) Present studyN. mexicanus PE Mexico H7 / HQ285426 Present studyN. mexicanus PE Mexico H7 / HQ285426 Present studyN. mexicanus PE Mexico H7 / HQ285426 (EBD28508) Present studyN. mexicanus PE Mexico H7 / HQ285426 (EBD28510) Present studyN. mexicanus PE Mexico H8 / HQ285427 (EBD28507) Present studyN. mexicanus PE Mexico H8 / HQ285427 Present studyN. mexicanus SA Mexico H7 / HQ285426 (EBD28506) Present studyN. mexicanus SA Mexico H7 / HQ285426 Present studyN. mexicanus SA Mexico H7 / HQ285426 (EBD28509) Present studyN. mexicanus SA Mexico H8 / HQ285427 Present studyN. mexicanus TR Mexico H9 / HQ285428 Present studyN. mexicanus TR Mexico H10 / HQ285429 Present studyN. mexicanus JI Mexico H11 / HQ285430 Present studyN. mexicanus JI Mexico H11 / HQ285430 Present studyN. mexicanus JI Mexico H11 / HQ285430 Present studyN. mexicanus TG Mexico H12 / HQ285434 Present studyN. mexicanus TG Mexico H12 / HQ285434 Present studyN. mexicanus TG Mexico H12 / HQ285434 Present studyN. mexicanus TG Mexico H12 / HQ285434 Present studyN. mexicanus TG Mexico H13 / HQ285435 Present studyN. mexicanus SS Mexico H14 / HQ285438 Present studyN. mexicanus SS Mexico H15 / HQ285439 Present studyN. mexicanus SS Mexico H15 / HQ285439 Present studyN. mexicanus SS Mexico H16 / HQ285440 Present studyN. mexicanus SS Mexico H17 / HQ285441 Present studyN. mexicanus CA Mexico H2 / HQ285421 Present studyN. mexicanus CA Mexico H2 / HQ285421 Present studyN. mexicanus CA Mexico H2 / HQ285421 Present studyN. mexicanus CA Mexico H2 / HQ285421 Present studyN. mexicanus CA Mexico H18 / HQ285442 Present studyN. mexicanus CA Mexico H19 / HQ285443 Present study


APPENDIX. Continued

Species Location Country Cyt b haplotypes / Genbank(voucher) Reference

Natalus mexicanus CA Mexico H20 / HQ285444 Present studyN. mexicanus CA Mexico H20 / HQ285444 Present studyN. mexicanus CAN Mexico H21 / JN244834 (RLW1994) Present studyN. mexicanus CAN Mexico H9 / HQ285428 (RLW1999) Present studyN. mexicanus CAN Mexico H22 / JN244835 (RLW1998) Present studyN. mexicanus CAN Mexico H22 / JN244835 (RLW1997) Present studyN. mexicanus CAN Mexico H23 / JN244836 (RLW2000) Present studyN. mexicanus CAN Mexico H9 / HQ285428 (RLW2001) Present studyN. mexicanus CAN Mexico H9 / HQ285428 (RLW1996) Present studyMyotis velifer Sonora Mexico AF376870 Ruedi and Mayer (2001),

Hoofer et al. (2003)Chilonatalus micropus St. Catherine Jamaica AY621026 Dávalos (2005),

Hoofer et al. (2003)Natalus major María Trinidad Dominican Republic AY621021 Dávalos (2005)N. jamaicensis St. Catherine Jamaica AY621023 Dávalos (2005)N. tumidirostris St. Andrew Trinidad AY621009 Dávalos (2005)

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