filogenetica molecular myotis

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Molecular phylogenetics of Myotis indicate familial-level divergence for the genusCistugo (Chiroptera)Author(s): Justin B. Lack , Zachary P. Roehrs , Craig E. Stanley Jr , Manuel Ruedi , and Ronald A. VanDen BusscheSource: Journal of Mammalogy, 91(4):976-992. 2010.Published By: American Society of MammalogistsDOI: http://dx.doi.org/10.1644/09-MAMM-A-192.1URL: http://www.bioone.org/doi/full/10.1644/09-MAMM-A-192.1

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Molecular phylogenetics ofMyotis indicate familial-level divergence forthe genus Cistugo (Chiroptera)

JUSTIN B. LACK,* ZACHARY P. ROEHRS, CRAIG E. STANLEY, JR., MANUEL RUEDI, AND RONALD A. VAN DEN BUSSCHE

Department of Zoology, 430 Life Sciences West, Oklahoma State University, Stillwater, OK 74078, USA (JBL, ZPR, CES,

RAVDB)

Department of Mammalogy and Ornithology, Natural History Museum, P.O. Box 6434, 1211 Geneva 6, Switzerland (MR)

* Correspondent: [email protected]

The genus Myotis has undergone significant taxonomic revision since the advent of DNA sequencing

techniques. Prior morphological examination of Myotis has indicated as many as 4 subgenera correlated with

foraging strategies. Recent studies using mitochondrial DNA (mtDNA) sequence data have questioned the

validity of these subgenera and have indicated that several taxa may require reevaluation as to their position

within Vespertilionidae. Nevertheless, no study has used large-scale nuclear DNA sequencing to examine

relationships within Myotis. We generated 4,656 base pairs (bp) of nuclear intron (PRKC1, STAT5A, and THY)

and exon (APOB, DMP1, and RAG2) sequence data in addition to 2,866 bp of mtDNA sequence data to test

previously hypothesized subgeneric groupings of Myotis. We included 21 species of Myotis from all

morphological subgenera previously suggested, representatives of all subfamilies and tribes currently

recognized in Vespertilionidae, and multiple representatives of all other families currently included in the

superfamily Vespertilionoidea. We also included a representative of the rare African genus Cistugo, because

significant doubt exists about its familial position. Our phylogenetic analyses did not support the

morphologically defined Myotis subgenera and confirm that morphological similarities among Myotis are the

result of convergent evolution. Divergence estimates derived from the total data set were concordant with

previous studies, suggesting a middle Miocene trans-Beringian dispersal from Asia colonized North America,

with subsequent South American colonization and diversification prior to the formation of the Isthmus of

Panama 34 million years ago. Myotis latirostris fell outside of Myotis, and the high genetic distance separating

it from other Myotis suggested that M. latirostris represented a distinct genus. The genus Cistugo, previously a

subgenus within Myotis, fell basal to all vespertilionids, with a high genetic distance separating it from

Vespertilionidae. We conclude that Cistugo should constitute a distinct family within Vespertilionoidea.

DOI: 10.1644/09-MAMM-A-192.1.

Key words: Cistugo, mitochondrial DNA, molecular dating, Myotis, nuclear DNA, phylogenetics

E 2010 American Society of Mammalogists

Conflicts between morphological and molecular data are

abundant in the literature (Lee 2001; Patterson 1987), and

evolutionary relationships within the order Chiroptera are no

exception. Using mitochondrial DNA (mtDNA) sequence

data, Ruedi and Mayer (2001) revealed significant convergent

evolution within Myotis that led them to propose a convergent

evolutionary hypothesis for morphological similarities. In

resolving higher-level chiropteran relationships and elucidat-

ing evolutionary origins of echolocation, Eick et al. (2005)

found that the majority of morphological characters they

examined were homoplastic and therefore taxonomically

misleading. As is evident from these studies, diverse data sets

are often necessary to obtain robust and accurate assessments

of evolutionary relationships.

The chiropteran family Vespertilionidae is the 2nd most

speciose mammalian family, with approximately 407 species

and an essentially worldwide distribution (Simmons 2005).

Because of extensive convergent evolution, lack of taxonom-

ically informative characters, and likely a rapid initial

diversification, resolving evolutionary relationships within

Vespertilionidae, based on morphology, has been difficult.

Since the advent of DNA sequencebased phylogenetic

techniques, significant taxonomic revisions have been made

to Vespertilionidae, with many of the morphology-based

w w w . m a m m a l o g y . o r g

Journal of Mammalogy, 91(4):976992, 2010

976

phylogenies modified (Hoofer and Van Den Bussche 2003).

Some of the most significant taxonomic changes included

elevation of the subfamily Miniopterinae to family status

(MiniopteridaeHoofer and Van Den Bussche 2003; Miller-

Butterworth et al. 2007), reorganization and elevation of the

tribe Myotini to subfamilial status (Myotinae), and identifica-

tion of many problematic assemblages at multiple taxonomic

levels (e.g., nonmonophyly of the genus Eptesicus and validity

of several tribes within Vespertilioninae).

The genus Cistugo (Thomas, 1912) consists of 2 species

endemic to southern Africa: C. seabrae and C. lesueuri

(Nowak 1999; Simmons 2005). Initial treatments of Cistugo

placed it in the subfamily Vespertilioninae as a distinct genus.

Nevertheless, the majority of classifications subsequent to

Thomas (1912) relegated Cistugo to subgeneric status within

Myotis (Corbet and Hill 1991; Ellerman and Morrison-Scott

1951; Hayman and Hill 1971; Koopman 1993, 1994). Recent

systematic studies using molecular and karyotypic data have

been interpreted as supporting full generic rank of Cistugo

(Bickham et al. 2004; Eick et al. 2005; Rautenbach et al. 1993;

Stadelmann et al. 2004), but due to a lack of sufficient

taxonomic sampling, it has remained uncertain whether

Cistugo represents the ancestral lineage within Vespertilioni-

dae or, alternatively, if Cistugo represents a distinct family

within Vespertilionoidea (Stadelmann et al. 2004).

Not only have the phylogenetic affinities between Cistugo

and Myotis been problematic, but Myotis itself has been a

source of significant systematic confusion. This genus is

composed of approximately 100 species, distributed across all

continents except Antarctica (Simmons 2005) and displays

high levels of ecological and behavioral diversity. Original

designations based on morphology proposed 3 subgeneric

groupings (Myotis, Leuconoe, and Selysius) corresponding to

modes of flight and feeding guilds (Findley 1972). Although

these subgeneric groupings have been challenged based on

dental characters (Godawa Stormark 1998; Menu 1987), many

classifications of vespertilionid bats continue to recognize

these subgenera. Koopman (1993, 1994) added Cistugo to the

subgenera of Findley (1972), thus producing 4 subgeneric

groupings. Multiple studies using DNA sequences have shown

that these subgenera are not natural assemblages that reflect

evolutionary history; instead, the phylogenetic relationships

reflect biogeographic relationships, with major clades corre-

sponding to continental landmasses and regions therein

(Hoofer and Van Den Bussche 2003; Ruedi and Mayer

2001; Stadelmann et al. 2004, 2007). Adding to the difficulties

in revealing the evolutionary history of Myotis, in the most

recent systematic assessment, M. latirostris fell basal to the

Myotis clade (although with relatively weak support),

indicating that this species might represent either the oldest

extant lineage of Myotis or even a distinct genus (Stadelmann

et al. 2007). With 1 exception, previous phylogenetic studies

of Myotis used only mtDNA sequences. Stadelmann et al.

(2007) analyzed 1,148 base pairs (bp) of the nuclear RAG2

gene; however, RAG2 produced few phylogenetically infor-

mative characters, therefore adding little to the mtDNA

phylogeny. Given the contrasting evolutionary relationships of

Cistugo, Myotis, and M. latirostris based on morphology and

mitochondrial sequence data, additional insight from nuclear

sequence data could clarify these relationships and help

stabilize the taxonomy of Vespertilionidae.

Because the majority of recent studies support elevation of

Cistugo to generic status but were unable to determine

whether Cistugo should be treated as a member of Vesperti-

lionidae or as a member of a distinct family within

Vespertilionoidea (Bickham et al. 2004; Eick et al. 2005;

Stadelmann et al. 2004), our 1st objective was to test the

validity of the Myotis subgenera Leuconoe, Myotis, and

Selysius as defined by Findley (1972) and the validity of

biogeographical grouping of species based on mtDNA

sequence data (Bickham et al. 2004; Ruedi and Mayer 2001;

Stadelmann et al. 2004, 2007). Our 2nd objective was to assess

the phylogenetic hypothesis that Cistugo represents a distinct

family within Vespertilionoidea. Additionally, Stadelmann et

al. (2007) suggested that M. latirostris represented either the

basal lineage of Myotis or possibly a distinct vespertilionid

genus. Therefore, our 3rd objective was to assess the

phylogenetic hypotheses of Stadelmann et al. (2007) regarding

the uniqueness of M. latirostris. Although the questions

associated with our objectives have been examined in several

recent molecular studies (Bickham et al. 2004; Ruedi and

Mayer 2001; Stadelmann et al. 2004, 2007), all of those

studies examined portions of the mitochondrial genome.

Because of its maternal inheritance and therefore differential

accumulation of mutations relative to the nuclear genome,

phylogenetic analysis of mtDNA only can result in a

phylogeny not indicative of the true evolutionary history for

a given taxon (Avise 1994). To address our 3 objectives, we

generated DNA sequence data from 3 nuclear exons (APOB,

DMP1, and RAG2) and 3 nuclear introns (PRKC1, STAT5A,

and THY) from a taxonomically diverse sampling of

Vespertilionoidea and combined these nuclear DNA sequence

data with mitochondrial ribosomal DNA sequences previously

generated for these same taxa (Hoofer and Van Den Bussche

2003; Van Den Bussche and Hoofer 2004).

MATERIALS AND METHODS

We included 80 ingroup taxa representing Vespertilionidae,

Molossidae, Miniopteridae, and Natalidae, the 4 families that

molecular studies have indicated comprise the superfamily

Vespertilionoidea (Eick et al. 2005; Teeling et al. 2005; Van

Den Bussche and Hoofer 2004). Within Vespertilionidae, we

included representatives of all vespertilionid subfamilies and

tribes indicated in Hoofer and Van Den Bussche (2003; see

Appendix I). Finally, we included representatives of Embal-

lonuridae, Phyllostomidae, Mormoopidae, Noctilionidae,

Thyropteridae, and Myzopodidae as outgroup taxa.

Total genomic DNA was extracted from heart, liver, kidney,

or muscle tissue samples following standard protocols (Long-

mire et al. 1997). Three nuclear exons (apolipoprotein b

[APOB], dentin matrix acidic phosphoprotein 1 [DMP1], and

August 2010 LACK ET AL.MOLECULAR PHYLOGENETICS OF MYOTIS 977

recombination activating gene 2 [RAG2]) and 3 nuclear

introns (protein kinase C iota [PRKC1], signal transducer and

activator of transcription 5A [STAT5A], and thyrotropin

[THY]) were targeted using previously designed primers

(Appendix II). Polymerase chain reaction amplifications were

carried out in 30-ml reactions containing 200500 ng of DNA,0.14 mM of each deoxynucleoside triphosphate, 6 ml of 103buffer, 3.5 mM of MgCl2, 0.8 mg/ml of bovine serum

albumin, 0.15 mM of each primer, 1 unit Taq polymerase, anddouble distilled water to volume. The general thermal profile

consisted of an initial denaturation of 94uC for 3 min, followedby 35 cycles of 94uC for 1 min, annealing for 1 min (seeAppendix II for annealing temperatures), and 72uC for12 min. A final elongation of 72uC for 30 min ensured allreactions ran to completion. In addition, we amplified the 12S

rRNA, tRNAVal, and 16S rRNA regions of the mitochondrial

genome for taxa not previously examined following the

methods of Van Den Bussche and Hoofer (2000). Double-

stranded products were purified using the Wizard SV Gel PCR

Prep DNA Purification System (Promega, Madison, Wiscon-

sin), and both strands of the purified polymerase chain

reaction products were sequenced using Big Dye 1.1 chain

terminators and an ABI 3130 Genetic Analyzer (Applied

Biosystems, Inc., Foster City, California).

The mitochondrial and 6 nuclear data sets were aligned

independently using Clustal X software (Thompson et al.

1997), and the resulting multiple alignments were imported

into MacClade (Maddison and Maddison 2000) where each

alignment was visually inspected and manually optimized. All

of the nuclear alignments and the mitochondrial data

contained insertiondeletion (indel) events, and gaps were

introduced (either by Clustal X or manually) to optimize the

alignment. All indel regions were examined carefully, and any

position in the alignment where character state was not

assigned confidently for all taxa was excluded from all

analyses. Pairwise genetic distances were calculated in

PAUP* (Swofford 2003) using maximum-likelihood (ML)

model parameters estimated by MODELTEST version 3.06

(Posada and Crandall 1998).

A likelihood mapping analysis (Strimmer and von Haeseler

1997) as implemented in TREEPUZZLE 5.2 was conducted to

quantify and compare the phylogenetic signal for the mtDNA

and concatenated nuclear data sets. Incongruence between

individual genetic markers (i.e., mtDNA versus RAG2, etc.)

was determined by running maximum-parsimony (MP), ML,

and Bayesian phylogenetic analyses for each data set

independently and employing the 90% conflict criterion (DeQueiroz 1993). This revealed no conflicting, well-supported

nodes among data sets (well-supported nodes requiring 70%bootstrap support for MP and ML analyses and 0.95posterior probability for Bayesian analyses). For further

analyses we used 2 data sets: combined nuclear data and

nuclear and mtDNA combined. Representatives of Phyllosto-

midae, Myzopodidae, Thyropteridae, Mormoopidae, Noctilio-

nidae, and Emballonuridae were selected to serve as out-

groups. We did not analyze the mtDNA data set separately

because the data set differed from that analyzed by Hoofer and

Van Den Bussche (2003) by only a few taxa. Analyses of the

nuclear and combined nuclear and mitochondrial data sets

were conducted as described below.

Maximum-likelihood and MP methods were carried out

using PAUP* (Swofford 2003), whereas Bayesian phyloge-

netic analyses were conducted using MrBayes version 3.1.2

(Huelsenbeck and Ronquist 2001). For ML analyses the most

appropriate model of nucleotide substitution was evaluated

using MODELTEST version 3.06 (Posada and Crandall 1998),

and trees were constructed using nearest neighbor interchange

branch-swapping. For MP analyses, trees were generated

using equal weighting and the heuristic search option, the

maximum number of trees retained set at 500, tree-bisection-

reconnection branch-swapping, and 25 random additions of

input taxa. Reliability of clades from the ML and MP analyses

was evaluated via bootstrap analysis. For the ML analyses, we

performed 100 iterations with a heuristic search and nearest

neighbor interchange branch-swapping. For the MP bootstrap

analysis, we performed 1,000 iterations with a heuristic search

that included 25 random additions of taxa and tree-bisection-

reconnection branch-swapping. Bayesian analyses were per-

formed using 4 simultaneous Markov chains run for 5 3 106

generations, with random, unconstrained starting trees. The

analyses employed the GTR + I + C model of nucleotidesubstitution; values for model parameters were not defined a

priori but were treated as unknown variables with uniform

priors. Trees were sampled every 100 generations and

temperature was set at 0.02. Resulting burn-in values (the

point at which the model parameters and tree scores reached

stationarity) were determined empirically by evaluating

likelihood scores. All runs were checked for sufficient mixing,

stable convergence on a unimodal posterior, and effective

sample sizes (Drummond et al. 2002) . 100 for all parametersusing TRACER version 1.4 (Drummond and Rambaut 2003).

For the combined data set we estimated node ages using the

Markov chain Monte Carlo sampling method implemented in

the program BEAST version 1.4.8 (Drummond and Rambaut

2007). To compare the null molecular clock model versus the

alternative model, in which each branch is allowed its own

unique rate, we used the likelihood ratio test (Felsenstein

1981). In the Bayesian framework Bayes factors (Kass and

Raftery 1995; Newton and Raftery 1994; Suchard et al. 2001),

as implemented in the program TRACER version 1.4

(Drummond and Rambaut 2003), were used to compare the

relaxed uncorrelated exponential clock and relaxed uncorre-

lated lognormal clock. These analyses used the GTR + I + Cmodel of sequence evolution and a Yule speciation tree prior

(Yule 1924). Fossil calibrations were used to place a prior on 2

nodes. A minimum age of 30 million years ago (mya) was

used for the PhyllostomidaeMormoopidae divergence with a

uniform distribution (Teeling et al. 2005) because the oldest

fossils uniting this group are found in the Whitneyan 30

32 mya (G. S. Morgan, University of Florida, pers. comm.).

Therefore, the maximum age of the MormoopidaePhyllosto-

midae divergence (the maximum of the uniform distribution)

978 JOURNAL OF MAMMALOGY Vol. 91, No. 4

was set at the EoceneOligocene boundary (34 mya), because

actual divergences occurred some time before the fossil

formed. The 2nd calibration was a minimum of 37 mya for the

split between Vespertilionidae and Molossidae (Teeling et al.

2005), because verified vespertilionid and molossid fossils

have been found from the middle Eocene (McKenna and Bell

1997). We used a lognormal prior distribution (offset 5 37.0,X 5 0, SD 5 1.3) on this calibration to encapsulate the entiremiddle Eocene in the prior.

Using the above calibrations as point estimates, we

generated a chronogram using r8s version 1.60 (Sanderson

2003) to provide a starting tree for the dating analysis. This

was done by allowing r8s to rescale the branch lengths on the

phylogram resulting from phylogenetic analysis of the

concatenated nuclear and mitochondrial sequence data to time

rather than substitutions per site. For the BEAST analysis an

initial run of 30,000,000 generations with 10% burn-in wasrun to optimize operators. The final analysis consisted of 2

separate runs of 30,000,000 generations, each with 10% burn-in. Results of these final 2 runs were log combined to obtain

final estimates of divergence, and all runs were checked for

sufficient mixing, stable convergence on a unimodal posterior,

and effective sample sizes (Drummond et al. 2002) . 100 forall parameters using TRACER version 1.4 (Drummond and

Rambaut 2003).

RESULTS

All sequences generated in this study were submitted to

GenBank (see Appendix I for accession numbers), and each

alignment is available from TreeBase (http://treebase.org).

Results of the likelihood-mapping analysis indicated phylo-

genetic signal was nearly identical for the mtDNA and nuclear

data sets with 94.9% and 96.0% of quartets resolved,respectively. Phylogenetic analyses of the separate nuclear

and combined data sets revealed few conflicting supported

nodes. All MP and ML bootstrap proportions are referred to as

MPBS and MLBS, respectively, and Bayesian posterior

probabilities are referred to as PP.

Molecular Results

Nuclear analyses.Concatenation of the APOB, DMP1,

PRKC1, RAG2, STAT5A, and THY DNA sequences for each

taxon resulted in a total of 4,656 aligned positions. Because of

potential violation of the assumption of positional homology,

513 positions were excluded from phylogenetic analyses. Of the

remaining 4,143 positions, 2,352 were variable, and 1,592 were

parsimony informative. The likelihood ratio test implemented

in MODELTEST indicated the GTR model of evolution with a

proportion of invariant sites (I) and gamma distributed among-

site rate variation (C) was most appropriate. For the combinednuclear ML analysis, model parameter values were set to those

estimated by MODELTEST and were as follows: I 5 0.1278;shape parameter a of the gamma distribution 5 1.2077; base

frequencies 5 0.2767, 0.2384, 0.2431, 0.2418; R-matrix 51.1001, 3.1682, 0.7205, 0.7205, 3.8490.

Maximum-parsimony analysis of the concatenated nuclear

sequences resulted in 2 equally parsimonious trees of 7,126

steps (consistency index [CI]5 0.4598, retention index [RI]50.7092), and the ML analysis resulted in a single optimal tree

(lnL 5 42,034.92810; Fig. 1). The nuclear Bayesian analysisreached stationarity at approximately 185,000 generations, so

only trees following a conservative burn-in of 200,000

generations were analyzed. All parameters sampled in the

Bayesian analysis converged on a stable, unimodally distrib-

uted posterior, indicating mixing was sufficient. ML and

Bayesian phylogenies were identical and differed from a

nuclear MP consensus phylogeny only in the placement of

Miniopteridae and of the long branch leading to Eptesicus

dimissus. MP analysis resulted in the miniopterids being sister

to the molossids, with moderate support (70% MPBS), andML and Bayesian phylogenies resulted in the miniopterids

being sister to Vespertilionidae, with moderate support (76%MLBS, 0.97 PP). Because statistical support (albeit slight) was

higher for the MiniopteridaeVespertilionidae sister relation-

ship, that relationship is shown in Fig. 1. ML and Bayesian

analyses placed E. dimissus basal to the genus Pipistrellus,

with significant statistical support (70% MLBS, 1.0 PP) andnot with other species of Eptesicus. MP analysis placed E.

dimissus sister to Hypsugo cadornae (90% MPBS), with thatpair then being basal to bats in the tribes Nycticeiini,

Pipistrellini, and Vespertilionini (sensu Hoofer and Van Den

Bussche 2003) with strong support (89% MPBS).Combined nuclear and mitochondrial analyses.The

combined nuclear and mitochondrial data set resulted in

7,522 aligned positions. Because of potential violation of the

assumption of positional homology, 1,339 positions were

excluded from phylogenetic analyses. Of the remaining 6,183

aligned positions, 3,388 were variable, and 2,454 were

parsimony informative. The likelihood ration test implement-

ed in MODELTEST indicated the GTR + I + C model ofnucleotide substitution most appropriately fit our data. For the

ML analysis model parameter, values were: I 5 0.2420; a 50.6458; base frequencies 5 0.3239, 0.2041, 0.2191, 0.2529;R-matrix 5 1.4108, 4.3556, 0.8461, 1.0084, 8.2028.

Maximum parsimony analysis resulted in 10 equally

parsimonious trees of 12,864 steps (CI 5 0.3275, RI 50.6360) and the ML analysis resulted in a single optimal tree

(lnL5 75,061.54406; Fig. 2). The Bayesian analysis reachedstationarity at approximately 180,000 generations, so only

trees following a conservative burn-in of 200,000 generations

were analyzed. All parameters sampled in the Bayesian

analysis converged on a stable, unimodally distributed

posterior, indicating mixing was sufficient. The ML and

Bayesian phylogenies of the combined nuclear and mitochon-

drial data were identical and differed from an MP consensus

phylogeny only in the placement of Miniopteridae. Again,

Bayesian and ML statistical support (76% MLBS, 0.95 PP,respectively) for the MiniopteridaeVespertilionidae sister

relationship was slightly higher than that for the Miniopter-


idaeMolossidae sister relationship returned in the MP

analysis (70% MPBS). As a result, only the MLBayesianphylogeny is shown (Fig. 2).

Molecular dating.The likelihood ratio test significantly

rejected the molecular clock for the combined data set (2DL5118.647; P 5 0.002). For Bayes factors, a value of logeB10 .2 is taken to be positive support for the alternative model (in

this case, the relaxed uncorrelated lognormal clock), and a

logeB10 .10 indicates very strong support. Bayes factorsindicated that the relaxed uncorrelated lognormal clock was

favored over the relaxed uncorrelated exponential clock

(logeB10 5 4.822). However, because this value was not.10, we conducted dating analyses using both the relaxeduncorrelated exponential clock and the relaxed uncorrelated

lognormal clock. Divergence values for the 2 models were

nearly identical, with differences growing slightly as estimates

approached the root. Because of the overall concordance

between these 2 analyses and the slight favoring of the

lognormal model according to Bayes factors, only the results

of the relaxed uncorrelated lognormal clock analysis are

shown (Fig. 3).

We estimate the age of divergence between the Old World

and New World Myotis to have occurred approximately

13.4 mya (Fig. 3). The time to most recent common ancestor

for the Old World clade was approximately 10.9 mya and for

the New World clade was approximately 9.1 mya. Within the

New World clade, neotropical and Nearctic Myotis radiated

approximately 7.5 mya.M. latirostris diverged from the rest of

Myotis approximately 18 mya, and Cistugo diverged from all

other vespertilionids 34 mya. The time to most recent common

ancestor for Vespertilionidae was 27.1 mya. Within Vesperti-

lioninae (sensu Hoofer and Van Den Bussche 2003),

divergence estimates should be approached with caution,

because many of the intertribal relationships were unresolved

in our analyses.

Taxonomic Results

Cistugidae, new family

Type genus.Cistugo Thomas, 1912.

Diagnosis, description, and comparisons.Description of

this new family largely follows the description of Cistugo by

Thomas (1912). Members of Cistugidae are small, pipistrelle-

like bats (forearm length 3236 mm; mass approximately 4 g)

and possess 24 glands in the wing plagiopatagium just

posterior to the forearm (Seamark and Kearney 2006:figs. 4

FIG. 1.Maximum-likelihood phylogram (2lnL 5 42,034.92810) based on the GTR + I + C substitution model for the concatenated nucleardata set. The number of asterisks indicates support by 1 (*), 2 (**), or all 3 (***) phylogenetic inference methods. Clades are considered

supported when bootstrap proportions are 70% or the Bayesian posterior probability is 0.95, or both. Outgroup taxa and representatives ofnon-Myotinae vespertilionids, Molossidae, and Natalidae were reduced to single branches for presentation. See Appendix I for specific taxa.


and 5) that are not present in any other vespertilionoid bat.

These glands have been described as reduced in C. lesueuri or

even absent from museum specimens (Smithers 1983), but the

wing glands typically become apparent when dried specimens

are wetted (Herselman and Norton 1985; Roberts 1951;

Shortridge 1942). Members of Cistugidae possess 38 teeth,

similar to Myotis, with 2 pairs of small and 1 pair of larger

premolars on each jaw (Roberts 1951:plate 10); lower molars

are myotodont (Menu and Sige 1971). Species in Cistugidae

lack distinguishing dental morphology compared with Myotis,

and therefore Vespertilionidae, but given the nondescript

nature of Vespertililonidae (Koopman 1994; Tate 1942), this

is not surprising.

Cytogenetically, species in Cistugidae have a diploid

number of 2n 5 50 chromosomes and are completely distinctfrom all Myotis (2n 5 44Rautenbach et al. 1993) and othervespertilionoids (Natalidae, 2n 5 36; Molossidae, 2n 5 48;Miniopteridae, 2n 5 46; Vespertilionidae, 2n 5 2658Baker and Jordan 1970; Volleth and Heller 1994; Zima and

Horacek 1985), except for Eptesicus (2n 5 50Bickham etal. 2004). Eick et al. (2005) cited the presence of multiple

indels in 2 introns (SPTBN and PRKC1) that further

distinguish species of Cistugidae from Vespertilionidae. We

also recovered a single 18-bp indel in exon 6 of the DMP1

gene not present in any other species of Vespertilionoidea we

examined.

Geographical distribution.Currently restricted to southern

Africa south of 15uS latitude. The distribution of C. lesueuri isknown from scattered records from across South Africa in

northern portions of Western Cape and southeastern Northern

Cape, eastern Free State, and Lesotho (Herselman and Norton

1985; Lynch 1994; Watson 1998). C. seabrae is distributed

from southwestern Angola through western Namibia and into

the northern portion of the Namakwa District, and western

portions of the Siyanda District of Northern Cape Province,

South Africa (Seamark and Kearney 2006; Smithers 1983).

Etymology.Cistugidae is derived from the genus Cistugo,

and the family ending -idae (Article 29, International Code of

FIG. 2.Maximum-likelihood phylogram (2lnL 5 75,061.54406) based on the GTR + I + C substitution model for the concatenated nuclearand mitochondrial DNA data set. The number of asterisks indicates support by 1 (*), 2 (**), or all 3 (***) phylogenetic inference methods.

Clades are considered supported when bootstrap proportions are 70% or the Bayesian posterior probability is 0.95, or both. Outgroup taxaand representatives of non-Myotinae vespertilionids, Molossidae, and Natalidae were reduced to single branches for presentation. See Appendix

I for specific taxa.


Zoological NomenclatureInternational Commission on Zoo-

logical Nomenclature 1999). The etymology of the genus

Cistugo was not detailed by Thomas (1912).

DISCUSSION

Based on strong support in all phylogenetic methods, the

single representative species of Cistugo fell outside Vesperti-

lionidae, which was congruent with results from past

systematic studies (Bickham et al. 2004; Eick et al. 2005;

Stadelmann et al. 2004). As was apparent in both the nuclear

and combined nuclear and mitochondrial phylograms (Figs. 1

and 2), Cistugo was significantly divergent from Vespertilio-

nidae. Although this significant divergence of Cistugo from

Vespertilionidae has been noted previously, none of the

previous studies included sufficient representation of Vesper-

tilionoidea to evaluate if Cistugo represented a distinct family

or was the most basal lineage of Vespertilionidae. Because

previous studies have suggested that Cistugo may represent a

distinct family, we included representatives of Miniopteridae,

Molossidae, and Natalidae to evaluate distinctiveness of

Cistugo. Corrected estimates of ML divergence based on our

combined nuclear and mitochondrial data set (Table 1)

indicated an average divergence of 22.17% between Cistugoand Vespertilionidae. Cistugo was similarly diverged from the

other 2 most closely related vespertilionoid families, with

average pairwise divergences of 22.82% with Miniopteridaeand 20.16% with Molossidae. Good justification was obtained

FIG. 3.Chronogram resulting from the relaxed uncorrelated lognormal clock molecular dating analysis of the combined data set conducted

in BEAST version 1.4.8. Shaded bars represent the 95% highest posterior density interval for divergence estimates. Divergence estimatescorrespond to the mean node ages in units of millions of years before present. Noctilionidae, Molossidae, Phyllostomidae, Natalidae, and non-

Myotinae vespertilionid lineages were collapsed for presentation. See Appendix I for specific taxa.


from our molecular results alone, or in combination with data

from several other sources, for recognizing Cistugo as a family

distinct from Vespertilionidae.

Rautenbach et al. (1993) reported that the karyotype of

Cistugo contained an all-acrocentric autosomal complement

and possessed a 2n 5 50. That diploid number was differentfrom all Myotis species examined to date (Zima and Horacek

1985) but was shared with Eptesicus. As discussed by Bickham

et al. (2004), that Cistugo and Eptesicus share the same diploid

number can be explained by this karyotype being the ancestral

condition for vespertilionids, which was proposed originally by

Stock (1983). Morphologically, C. seabrae and C. lesueuri are

distinct from Vespertilionidae because they possess wing

glands of unknown function (Thomas 1912). Although M.

vivesi also possesses glandular structures on the wings, they are

relatively reduced and occur at the center of the wing; wing

glands of Cistugo are pronounced and nearer the forearm.

Although their taxonomic sampling of vespertilionoids was

insufficient to address the distinctness of Cistugo, Eick et al.

(2005) concluded thatCistugowas distinct from vespertilionids.

Based on their analysis of nuclear DNA sequences, Eick et al.

(2005) found that all vespertilionids they examined were

characterized by a unique deletion in the SPTBN intron that was

absent in Cistugo, and further, the 2 species of Cistugo had a

unique insertion in the PRKC1 intron that was not present in

any vespertilionids. Thus, based on the totality of evidence,

including karyotypic and morphologic data, DNA sequence

data from mitochondrial cytochrome-b, 12S rRNA, tRNAVal,

and 16S rRNA genes, and a combination of nuclear exons and

introns, Cistugo is different from Myotis, and possesses a level

of genetic distinctiveness from Vespertilionidae. Moreover, the

level of genetic distinctiveness is equal to, or greater than,

differences between Vespertilionidae and Miniopteridae or

Molossidae and differences between Phyllostomidae and

Mormoopidae. Therefore, we recognize Cistugo as constituting

a separate family within Vespertilionoidea and a sister taxon to

Vespertilionidae with Miniopteridae being sister to this group

(Figs. 1 and 2).

For Myotis, results of our combined nuclear and mitochon-

drial analyses were generally congruent with previous mtDNA

phylogenies (Bickham et al. 2004; Hoofer and Van Den

Bussche 2003; Kawai et al. 2004; Ruedi and Mayer 2001;

Stadelmann et al. 2004, 2007), with strongly supported

monophyletic clades corresponding to Old World (with the

exception of M. latirostris, addressed below) and New World

species (Fig. 2) and a collapse of the previously recognized

morphological subgenera. Within the Old World clade, no

statistically supported relationships conflicted between the

nuclear and combined nuclear and mitochondrial data sets.

The addition of nuclear data provided identical relationships to

those previously reported, although some previously unre-

solved relationships were resolved with the additional data.

The Mediterranean species M. capaccinii fell out basal to the

South Pacific species M. moluccarum and M. cf. browni, with

strong statistical support in all inference methods. For the

European M. myotis the combined nuclear and mitochondrial

data set did not resolve its position within the Old World

Myotis, but nuclear analyses alone placed it basal to the 2

African species, M. bocagii and M. welwitschii, with strong

statistical support. Unfortunately, we were unable to test the

hypothesized subclades that Ruedi and Mayer (2001) and

Stadelmann et al. (2004) suggested for the Old World because

our sampling of this group was too sparse.

Within the New World clade the Nearctic and neotropical

subclades previously outlined by Ruedi and Mayer (2001),

Hoofer and Van Den Bussche (2003), and Stadelmann et al.

(2007) were recovered (Fig. 2). Monophyly of these 2

subclades was supported only in the Bayesian analysis for

the combined nuclear and mitochondrial data set but in all 3

methods of the phylogenetic analysis of the nuclear data set.

Some conflict also existed between the 2 data sets concerning

relationships within the subclades. Two Nearctic sister

species, M. velifer and M. yumanensis, are nested within the

neotropical subclade with high support in the combined

nuclear and mitochondrial analyses. In the analysis of the

nuclear data set those 2 species were not sister to each other,

with the position of M. velifer unresolved and M. yumanensis

basal to the neotropical subclade with strong support from all

inference methods (100% MPBS and MLBS, 1.0 PP). Thisposition of M. yumanensis may have some historical

biogeographic implications. Stadelmann et al. (2007) suggest-

ed a Palearctic origin for ancestors of the New World Myotis

during the Miocene, with an initial crossing of the Bering

Strait followed by a southern radiation, eventually reaching

South America. Perhaps M. yumanensis represents the closest

extant relative of the Nearctic ancestor that gave rise to all

neotropical Myotis, and the more slowly evolving nuclear

markers still retain this phylogenetic signal.

Taxonomic conflict also existed for M. fortidens, which

occurs from southern Mexico into Guatemala (Simmons

2005). Biogeographically, this would place it in the neotrop-

ical group, a relationship that was supported in the analysis of

the combined nuclear and mitochondrial data set, although

statistically only by the Bayesian analysis (1.0 PP). For the

TABLE 1.Maximum-likelihood corrected pairwise differences among Cistugo and families represented in Figs. 2 and 3.

Taxon Cistugo Vespertilionidae (excluding Cistugo) Miniopteridae Natalidae Molossidae

Cistugo

Vespertilionidae (excluding Cistugo) 22.17%

Miniopteridae 22.82% 25.50%

Natalidae 19.13% 21.86% 19.71% Molossidae 20.16% 23.01% 20.69% 16.96%


nuclear data set, M. fortidens was nested within the Nearctic

clade, but that relationship was supported statistically only

with MP (82% MPBS). This species was not included in themore taxonomically dense mtDNA phylogenetic study of New

World Myotis, conducted by Stadelmann et al. (2007). Hoofer

and Van Den Bussche (2003) did include it in their study and

found strong support for its position nested within the

neotropical subclade. Our study, and that by Hoofer and Van

Den Bussche (2003), included ,50% of the described Myotisin the New World and a more thorough sampling of the 2 New

World subclades could provide clarification for the phyloge-

netic position of M. fortidens.

One notable exception to the monophyly of the Old World

Myotis was the East Asian M. latirostris, which was basal to

all Myotis in both data sets. Its position was supported by all

phylogenetic methods (Fig. 2). This species has been included

in only 1 other molecular phylogenetic study, and its results

also suggest, although with weak statistical support, that M.

latirostris might constitute a distinct genus (Stadelmann et al.

2007). We used corrected ML distances on the combined data

set to determine the extent of divergence between M.

latirostris and the remainder of Myotis. Corrected average

genetic distance between M. latirostris and all other Myotis

was 8.88%. The average pairwise divergence among allMyotis sampled here (excluding M. latirostris) was 6.29%.Average pairwise divergences among some other more

densely sampled genera included in this study were 4.19%,7.41%, 5.67%, 9.19%, and 4.36% for Scotophilus, Pipis-trellus, Eptesicus (excluding E. dimissus), Kerivoula, and

Miniopterus, respectively. Khan (2008) suggested the pres-

ence of multiple divergent lineages within Kerivoula based on

cytochrome-b sequence data, potentially corresponding to

multiple genera and explaining the high value obtained for that

genus. Further support for recognition of M. latirostris

belonging to a genus distinct from Myotis comes from

comparison of divergence values between closely related

genera. We found 9.18% divergence between the closelyrelated Harpiocephalus and Murina, a value comparable to

that found between M. latirostris and the remainder of Myotis

(8.88%). However, a more rigorous sampling of Myotis, morespecifically East Asian taxa, and comparative morphological

diagnosis are necessary to make a more thorough systematic

determination.

Molecular dating analyses returned divergence estimates for

Myotis highly concordant with those of past studies (Stadel-

mann et al. 2004, 2007), and estimates outside of Myotis also

were concordant with previous studies (Eick et al. 2005;

Teeling et al. 2005), indicating that divergences produced here

are robust. Our divergence estimates indicate that the split

between Old World and New World Myotis occurred during

the middle Miocene, approximately 13 mya. During much of

the Oligocene and early Miocene the tropical climate of the

Eocene gave way to modern temperate climates, with the

polar regions becoming dominated by ice. This transition

marked the most significant cooling event of the Cenozoic era

(Zanazzi et al. 2007) and was likely responsible for significant

faunal and floral shifts (Haines 1999). In addition, an abrupt

drop in sea level produced the Bering land bridge, allowing a

significant faunal exchange between Asia and North America

(Haines 1999; Wolfe 1994). In accordance with Stadelmann et

al. (2007), our evolutionary timescale also supports a middle

Miocene colonization of North America from Asia via the

Bering Strait and subsequent diversifications in the late

Miocene. Within the New World Myotis the divergence

between the Nearctic and neotropical clades occurred 10

11 mya and is highly congruent with past estimates

(Stadelmann et al. 2007). This suggests that the intervening

body of water separating North and South America was not a

substantial barrier for dispersal and that the formation of the

Isthmus of Panama 34 mya (Collins et al. 1996) likely had

little effect on distributions or diversification of Myotis.

For M. latirostris the dating analysis suggests a divergence

from all other Myotis approximately 18 mya, 5 million years

prior to the Old WorldNew World divergence. This is much

more distant from the base of the Myotis radiation than the

divergence date (approximately 13 mya) suggested by Stadel-

mann et al. (2007). This suggests that the climatic or geologic

events that led to the initial diversification of all other Myotis

are not likely the same as those contributing to the divergence of

M. latirostris from the Myotis lineage. The divergence estimate

for Cistugo also indicates an ancient divergence relative to the

base of Vespertilionidae. We estimate that Cistugo diverged

from the Vespertilionidae lineage approximately 34 mya,

almost 7 million years before the base of the vespertilionid

radiation (approximately 27 mya). This places the divergence of

Cistugo from Vespertilionidae near the OligoceneEocene

boundary and the end of an extended tropical climate. The base

of the vespertilionid radiation appears to have occurred in the

late Oligocene, coinciding with extensive climatic cooling and

dramatic shifts in sea level (Ogg et al. 2008; Zanazzi et al.

2007).

In conclusion, the overall concordance between the nuclear

and combined nuclear and mitochondrial data sets analyzed in

this study, coupled with the concordance of our results with

those of previous studies, indicate that the phylogenies

presented here provide an accurate and robust representation

of the true evolutionary history of these taxa. More

specifically, the deep divergence between Cistugo and the

vespertilionids indicates that this taxon represents a distinct

family (Cistugidae), sister to Vespertilionidae. The presence of

clades within Myotis that correspond to biogeographical

regions rather than to morphological or behavioral assem-

blages indicates, as originally suggested by Ruedi and Mayer

(2001), that convergent evolution has been extremely common

during the diversification of Myotis, and that Leuconoe,

Myotis, and Selysius are not valid as subgenera. This stands as

yet another example of habitat and environmental constraints

dictating evolutionary trajectories, illuminating the power of

natural selection in manipulating morphological and behav-

ioral diversity during a relatively rapid radiation. Finally, the

large amount of sequence divergence between M. latirostris

and all other Myotis indicates that this taxon most likely


represents a genus distinct from Myotis. Previous hypotheses

suggesting that Myotis originated in Asia and spread to its

current distribution (Findley 1972; Menu 1987) could be

substantiated with the phylogenetic position of the East Asian

M. latirostris. Divergence estimates support previously

hypothesized scenarios for the evolution of Myotis. In

addition, the divergence estimate for Cistugo from the

vespertilionid lineage lends further support that it is a taxon

distinct from vespertilionids, likely arising under different

paleoclimatic conditions than those to which basal vesperti-

lionids were exposed.

ACKNOWLEDGMENTS

We extend our sincere gratitude to S. E. Weyandt and M. Leslie for

assistance in the laboratory generating some of the RAG2 DNA

sequences during the initial stage of this project. We also thank G.

Eick for providing primer sequences and advice on polymerase chain

reaction profiles for amplification of some of the nuclear sequences.

For loaning tissue samples we extend our sincere gratitude to the

following persons and institutions, without whose generosity and

support this study would not have been possible: R. J. Baker of the

Natural Sciences Research Laboratory of the Museum of Texas Tech

University; N. B. Simmons of the American Museum of Natural

History; B. D. Patterson, L. R. Heaney, and W. T. Stanley of the Field

Museum of Natural History; S. B. McLaren of Carnegie Museum of

Natural History; M. D. Engstrom and B. Lim of the Royal Ontario

Museum; the late T. L. Yates of the Museum of Southwestern

Biology at the University of New Mexico; T. E. Lee, Jr., of Abilene

Christian University; J. O. Whitaker, Jr., and D. S. Sparks of the

Indiana State University Vertebrate Collection; R. L. Honeycutt and

D. Schlitter of the Texas Cooperative Wildlife Collection at Texas

A&M University; and P. J. Taylor of the Durban Natural Science

Museum. Thanks are also extended to D. S. Jacobs, C. Schoeman, and

B. Stadelmann, who helped collect Cistugo in South Africa. We also

thank the personnel of the Oklahoma State University Recombinant

DNA/Protein Resource Facility. Finally, we thank A. Gardner, R.

Pine, and 2 anonymous reviewers for their helpful comments that

made this a better manuscript. Financial support for this project was

provided by National Science Foundation grants DEB-9873657 and

DEB-0610844 to RAVDB and the G. & A. Claraz Foundation. Any

opinions, findings, and conclusions or recommendations expressed in

this material are those of the authors and do not necessarily reflect the

views of the National Science Foundation.

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Palaeogeography, Palaeoclimatology, Palaeoecology 108:207

216.

YULE, G. U. 1924. A mathematical theory of evolution, based on the

conclusions of Dr. J. C. Willis, F. R. S. Philosophical Transactions

of the Royal Society of London, B. Biological Sciences 213:2187.

ZANAZZI, A., M. J. KOHN, B. J. MACFADDEN, AND D. O. TERRY, JR.

2007. Large temperature drop across the EoceneOligocene

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ZIMA, J., AND I. HORACEK. 1985. Synopsis of karyotypes of

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Submitted 2 June 2009. Accepted 28 September 2009.

Associate Editor was Mark S. Hafner.


APPENDIXI

Specimensexamined.

Forcatalogued

specim

enswithmuseum

catalognumbers,

avoucher

ishousedin

collectionsat

American

Museum

ofNaturalHistory

(AMNH),Carnegie

Museum

ofNaturalHistory

(CM),Field

Museum

ofNaturalHistory

(FMNH),Museum

dH

istoireNaturelledeGene`ve(M

HNG),Museum

ofSouthwestern

Biologyat

theUniversity

of

New

Mexico(M

SB),Museum

ofTexas

TechUniversity

(TTU),OklahomaState

University

CollectionofVertebrates(O

SU),Royal

OntarioMuseum

(ROM),IndianaState

University

CollectionofVertebrates(ISUV),ortheUnited

StatesNational

Museum

ofNaturalHistory

(USNM).Foruncatalogued

specim

ens,only

atissuecollectionnumber

isgiven

andtissue

number

designationsareas

follows:

Texas

TechUniversity

tissue(TK),Carnegie

Museum

SpecialProject

tissue(SP),OklahomaState

University

tissue(O

K),Museum

dH

istoire

NaturelledeGene`vetissue(IZEA),Museum

ofSouthwestern

Biologyat

theUniversity

ofNew

Mexicotissue(N

K),Royal

OntarioMuseum

tissue(F),Rodney

L.Honeycuttpersonal

collection(RLH

and05M3),Durban

NaturalScience

Museum

(DM),AbileneChristianUniversity

tissue(A

CU),Manuel

Ruedipersonal

collection(M

),andDaleW.Sparkspersonal

catalog(D

WS).A

dash(

)denotesinform

ationunavailable

andtherefore

missing.GenBankaccessionnumbersforpreviouslypublished

sequencesareindicated

inboldface

type;

all

othersweregenerated

inthisstudy.

Taxon

Tissue

collectionno.

Museum

catalogno.

Locality

mtDNA

APOB

DMP1

RAG2

PRKC1

STAT5A

THY

Emballonuridae

Saccopteryxbilineata

AMNH267842

AMNH267842

Paracou,French

Guiana

AF263213

GU328198

AY141878

AY141015

AJ866288

AJ865391

AJ865636

Noctilionidae

Noctilio

albiventris

TK86633

BerbiceDistrict,

French

Guiana

AF263223

GU328180

AY141885

AF330811

AJ866308

AJ865413

AJ865658

Noctilio

leporinus

TK18515

CM63173

Saram

acca,

Surinam

e

AF263224

GU328181

AY141886

AF316477

AJ866309

AJ865314

AJ865659

Thyropteridae

Thyroptera

tricolor

AMNH268577

AMNH268577

Paracou,French

Guiana

AF263223

GU328207

AY141890

GU328118

GU328358

AJ865437

AJ865682

Morm

oopidae

Morm

oopsmegalophylla

TK78661

TTU79275

Barinas,Venezuela

AF263220

GU328151

AY141880

AY141020

AJ866302

AJ865406

GU328452

Phyllostomidae

Artibeusjamaicensis

TK4764

TTU35582

Guerrero,Mexico

AF263228

GU328132

AY141888

AF316444

AJ866315

AJ865420

AJ865665

Desmodusrotundus

AMNH267999

AMNH267999

Cayenne,

French

Guiana

AF263226

GU328123

GU328212

GU328048

AJ866314

AJ865419

AJ865664

Molossidae

Chaerephonpumilus

FMNH137634

FMNH137634

South

Buganda,

Uganda

AY495454

GU328126

GU328215

GU328051

GU328290

GU328365

GU328433

Otomopsmartiensseni

FMNH137633

FMNH137633

Burundi,Muramuya

AY495459

GU328185

GU328263

GU328097

GU328339

AJ865433

AJ865678

Sauromys

petrophilus

SP7791

CM105758

TransvaalProvince,

South

Africa

AY495460

GU328205

GU328282

GU328116

AJ866326

AJ865432

AJ865677

Myzopodidae

Myzopodaaurita

OK4246

USNM448885

Fianarantsoa,

Madagascar

AF345926

GU328147

AY141882

AY141022

GU328308

AJ865410

AJ865655

Natalidae

Natalusmicropus

TK9454

CM44578

Jamaica

AF345925

GU328179

AY141883

AY141023

AJ866307

AJ865412

AJ865657

Natalusstramineus

TK15660

TTU31457

St.John,Dominica

AF345924

GU328182

AY141884

AY141024

GU328337

GU328410

GU328480


Taxon

Tissue

collectionno.

Museum

catalogno.

Locality

mtDNA

APOB

DMP1

RAG2

PRKC1

STAT5A

THY

Miniopteridae

Miniopterusaustralis

TK20330

Central

Province,

PapuaNew

Guinea

AY395864

GU328148

GU328232

GU328066

AJ866297

AJ865401

AJ865646

Miniopterusfraterculus

TK33132

CM98058

RiftValley

Province,Kenya

AY495486

GU328149

GU328233

GU328067

AJ866298

AJ865402

AJ865647

Miniopterusinflatus

TK33539

CM98079

Western

Province,

Kenya

AY495487

GU328150

GU328234

GU328068

GU328309

GU328382

GU328451

Miniopterusschreibersii

TK40910

TTU70985

Beja,

Tunisia

AY395865

GU328153

GU328236

GU328070

GU328311

GU328384

GU328454

Miniopterustristis

TK20337

TTU36281

Central

Province,

PapuaNew

Guinea

AY495489

GU328154

GU328237

GU328071

GU328312

GU328385

GU328455

Vespertilionidae

Antrozouspallidus

NK506

California

GU328037

GU328120

GU328209

GU328045

GU328285

GU328360

GU328428

Antrozouspallidus

NK39195

Arizona

GU328038

GU328121

GU328210

GU328046

GU328286

GU328361

GU328429

Antrozouspallidus

TK49646

TTU71101

Texas

AF326088

GU328122

GU328211

GU328047

GU328287

GU328362

GU328430

Barbastella

barbastella

IZEA3590

MHNG1804.094

ValaisProvince,

Switzerland

AF326089

GU328124

GU328213

GU328049

GU328288

GU328363

GU328431

Bauerusdubiaquercus

F33200

ROM97719

Cam

peche,

Mexico

AY395863

GU328125

GU328214

GU328050

GU328289

GU328364

GU328432

Chalinolobusmorio

05M3

Australia

AY495462

GU328129

GU328218

GU328054

GU328292

GU328367

GU328435

Cistugoseabrae

M977

Goodhouse,South

Africa

GU328039

GU328127

GU328216

GU328052

AJ866334

AJ865443

AJ865688

Corynorhinusmexicanus

TK45849

Michoacan,Mexico

AF326090

GU328128

GU328217

GU328053

GU328291

GU328366

GU328434

Corynorhinusrafinesquii

TK5959

TTU45380

Arkansas

AF326091

GU328130

GU328219

GU328055

GU328293

GU328368

GU328436

Corynorhinustownsendii

OK11530

Oklahoma

AF263238

GU328131

AY141891

AY141029

GU328294

GU328369

GU328437

Eptesicusdiminutus

TK15033

TTU48154

Guarico,Venezuela

AY495465

GU328133

GU328220

GU328056

GU328295

GU328370

GU328438

Eptesicusdimissus

M1187

MHNG1926.053

PhongsaliProvince,

Lao

PDR

GU328040

GU328134

GU328221

GU328057

GU328296

GU328371

GU328439

Eptesicusfurinalis

AMNH268583

AMNH268583

Paracou,French

Guiana

AF263234

GU328135

GU328222

AY141030

GU328297

GU328372

GU328440

Eptesicusfuscus

SP844

CM102826

WestVirginia

AF326092

GU328136

GU328223

GU328058

GU328298

GU328373

GU328441

Eptesicushottentotus

TK33013

CM89000

RiftValley

Province,Kenya

AY495466

GU328137

GU328224

GU328059

AJ866329

AJ865438

AJ865683

Eudermamaculatum

NK36260

MSB121373

Utah

AF326093

GU328138

GU328225

GU328060

GU328299

GU328374

GU328442

Harpiocephalusharpia

TK21258

CM88159

Uthai

Thani

Province,Thailand

AF263235

GU328139

AY141892

AY141031

GU328300

GU328375

GU328443

Hypsugocadornae

M1183

MHNG1926.050

PhongsaliProvince,

Lao

PDR

GU328041

GU328140

GU328226

GU328061

GU328301

Not sequenced

GU328444

Hypsugonanus

DM7542

KwaZ

ulu-N

atal

Province,South

Africa

AY495474

GU328141

GU328227

GU328062

GU328302

GU328376

GU328445

Idionycterisphyllotis

ACU736

Utah

AF326094

GU328142

GU328228

GU328063

GU328303

GU328377

GU328446

Kerivoula

hardwickii

F44154

ROM110829

DongNai,Vietnam

AF345928

GU328143

AY141893

AY141034

GU328304

GU328378

GU328447

Kerivoula

lenis(analyzed

previouslyas

K.papillosa)

F44175

ROM110850

DongNai,Vietnam

AF345927

GU328144

GU328229

AY141035

GU328305

GU328379

GU328448

APPENDIXI.Continued.


Taxon

Tissue

collectionno.

Museum

catalogno.

Locality

mtDNA

APOB

DMP1

RAG2

PRKC1

STAT5A

THY

Kerivoula

pellucida

F35987

ROM102177

EastKalim

antan,

Indonesia

AY495476

GU328145

GU328230

GU328064

GU328306

GU328380

GU328449

Lasionycterisnoctivagans

TK24216

TTU56255

Texas

AF326095

GU328146

GU328231

GU328065

GU328307

GU328381

GU328450

Murinacyclotis

M1209

MHNG1926.034

PhongsaliProvince,

Lao

PDR

GU952767

GU328155

GU328238

GU328072

GU328313

GU328386

GU328456

Murinahuttoni

F42722

ROM107739

Dak

Lak,Vietnam

AY495490

GU328156

GU328239

GU328073

GU328314

GU328387

GU328457

Murinatubinaris

M1179

MHNG1926.034

PhongsaliProvince,

Lao

PDR

GU952768

GU328157

GU328240

GU328074

GU328315

GU328388

GU328458

Myotisalbescens

TK17932

CM77691

Marowijne,

Surinam

eAY495492

GU328159

GU328241

GU328076

GU328317

GU328390

GU328460

Myotisbocagii

FMNH150075

FMNH150075

TangaRegion,

Tanzania

AF326096

GU328160

GU328242

GU328077

GU328318

GU328391

GU328461

Myotiscf.browni

(analyzedpreviously

asM.muricola)

FMNH147067

FMNH147067

Mindanao

Island,

Philippines

AY495504

GU328169

GU328251

GU328086

GU328327

GU328400

GU328470

Myotiscalifornicus

TK78797

TTU79325

Texas

AY495495

GU328161

GU328243

GU328078

GU328319

GU328392

GU328462

Myotiscapaccinii

TK25610

TTU40554

NorthernProvince,

Jordan

AY495494

GU328162

GU328244

GU328079

GU328320

GU328393

GU328463

Myotisciliolabrum

TK83155

TTU78520

Texas

AY495497

GU328163

GU328245

GU328080

GU328321

GU328394

GU328464

Myotisdominicensis

TK15613

TTU31503

St.JosephParish,

Dominica

AY495500

GU328164

GU328246

GU328081

GU328322

GU328395

GU328465

Myotisfortidens

TK43186

Michoacan,Mexico

AY495502

GU328165

GU328247

GU328082

GU328323

GU328396

GU328466

Myotiskeaysi

TK13532

Yucatan,Mexico

AY495503

GU328166

GU328248

GU328083

GU328324

GU328397

GU328467

Myotislatirostris

M606

Moi-LiCounty,

Taiwan

GU952769

GU328167

GU328249

GU328084

GU328325

GU328398

GU328468

Myotislevis

FMNH141600

FMNH141600

Sao

Paulo,Brazil

AF326097

GU328168

GU328250

GU328085

GU328326

GU328399

GU328469

Myotismoluccarum

(analyzedpreviously

asM.adversus)

RLH62

Australia

AY495491

GU328158

Not sequenced

GU328075

GU328316

GU328389

GU328459

Myotismyotis

IZEA3790

MHNG1805.062

BernProvince,

Switzerland

AF326098

GU328170

GU328252

GU328087

GU328328

GU328401

GU328471

Myotisnigricans

FMNH129210

FMNH129210

Amazonas,Peru

AF326099

GU328171

GU328253

GU328088

GU328329

GU328402

GU328472

Myotisriparius

AMNH268591

AMNH268591

Paracou,French

Guiana

AF263236

GU328172

GU328254

GU328089

GU328330

GU328403

GU328473

Myotisseptentrionalis

DWS609

ISUV6454

Indiana

AY495507

GU328173

GU328255

GU328090

GU328331

GU328404

GU328474

Myotisthysanodes

TK78802

TTU79330

Texas

AF326100

GU328174

GU328256

GU328091

GU328332

GU328405

GU328475

Myotisvelifer

TK79170

TTU78599

Texas

AF263237

GU328175

GU328257

AY141033

GU328333

GU328406

GU328476

Myotisvolans

TK78980

TTU79545

Texas

AY495510

GU328176

GU328258

GU328092

GU328334

GU328407

GU328477

Myotiswelwitschii

FMNH144313

FMNH144313

KaseseDistrict,

Uganda

AY495511

GU328177

GU328259

GU328093

GU328335

GU328408

GU328478

Myotisyumanensis

TK28753

TTU43200

Oklahoma

AY495512

GU328178

GU328260

GU328094

GU328336

GU328409

GU328479

Nycticeinopsschlieffeni

TK33373

CM97998

Eastern

Province,

Kenya

AF326101

GU328183

GU328261

GU328095

AJ866330

AJ865440

AJ865685

Nycticeiushumeralis

TK26380

TTU49536

Texas

AF326102

GU328184

GU328262

GU328096

GU328338

GU328411

GU328481

Otonycterishem

prichii

SP7882

MaanGovernment,

Jordan

AF326103

GU328186

GU328264

GU328098

GU328340

GU328412

GU328482

Parastrellushesperus

TK78703

TTU79269

Texas

AY495522

GU328187

GU328265

GU328099

GU328341

GU328413

GU328483



Taxon

Tissue

collectionno.

Museum

catalogno.

Locality

mtDNA

APOB

DMP1

RAG2

PRKC1

STAT5A

THY

Perimyotissubflavus

TK90671

TTU80684

Texas

AY495523

GU328191

GU328269

GU328103

GU328345

GU328416

GU328487

Pipistrelluscoromandra

FMNH140377

FMNH140377

MalakandDivision,

Pakistan

AY495524

GU328190

GU328268

GU328102

GU328344

Not sequenced

GU328486

Pipistrellusjavanicus

FMMNH147069

FMMNH147069

Mindanao

Island,

Philippines

AY495525

GU328193

GU328271

GU328105

GU328347

Not sequenced

GU328489

Pipistrelluspygmaeus

(analyzedpreviously

asP.pipistrellus)

IZEA3403

MHNG1806.032

Barcelona

Province,Spain

AF326105

GU328195

GU328273

GU328107

GU328349

Not sequenced

GU328491

Plecotusauritus

IZEA2694

MHNG1806.047

ValaisProvince,

Switzerland

AF326106

GU328188

GU328266

GU328100

GU328342

GU328414

GU328484

Plecotusaustriacus

IZEA3722

MHNG1806.042

VaudProvince,

Switzerland

AF326107

GU328189

GU328267

GU328101

GU328343

GU328415

GU328485

Plecotusgaisleri

IZEA4780

MHNG1806.051

Morocco

GU328043

GU328192

GU328270

GU328104

GU328346

GU328417

GU328488

Plecotusmacrobullaris

IZEA4751

MHNG1806.053

ValaisProvince,

Switzerland

GU328044

GU328194

GU328272

GU328106

GU328348

GU328418

GU328490

Rhogeesa

parvula

TK20653

TTU36633

Sonora,Mexico

AF326109

GU328196

GU328274

GU328108

GU328350

GU328419

GU328492

Rhogeesa

tumida

TK40186

TTU61231

Valle,Honduras

AF326110

GU328197

GU328275

GU328109

GU328351

GU328420

GU328493

Scotophilusborbonicus

TK33267

CM98041

CoastalProvince,

Kenya

AY495532

GU328199

GU328276

GU328110

GU328352

GU328421

GU328494

Scotophilusdinganii

FMNH147235

FMNH147235

TangaRegion,

Tanzania

AY495533

GU328200

GU328277

GU328111

AJ866332

AJ865441

AJ865686

Scotophilusheathi

F42769

ROM107786

Dak

Lak,Vietnam

AY495534

GU328201

GU328278

GU328112

GU328353

GU328422

GU328495

Scotophiluskuhlii

FMNH145684

FMNH145684

Sibuyan

Island,

Philippines

AF326111

GU328202

GU328279

GU328113

GU328354

GU328423

GU328496

Scotophilusleucogaster

TK33359

CM90854

Eastern

Province,

Kenya

AY395867

GU328203

GU328280

GU328114

GU328355

GU328424

GU328497

Scotophilusnux

TK33484

Western

Province,

Kenya

AY495535

GU328204

GU328281

GU328115

GU328356

GU328425

GU328498

Scotophilusviridis

FMNH150084

FMNH150084

TangaRegion,

Tanzania

AF326112

GU328206

GU328283

GU328117

GU328357

GU328426

GU328499

Vespadelusregulus

RLH30

Australia

AY495539

GU328208

GU328284

GU328119

GU328359

GU328427

GU328500



APPENDIX II

Primer sequences, range of annealing temperatures, and reference for original primer sequences used to amplify and sequence the 6 nuclear

loci examined in this study. APOB, DMP1, and RAG2 are nuclear exons, whereas PRKC1, STAT5A, and THY are nuclear introns. Ex and In

refer to external and internal primers, respectively. F and R refer to forward and reverse primers, respectively.

Locus Primer name Primer sequence (59 to 39) Annealing temperature (uC) Reference

APOB APOBF GGCTGGACAGTGAAATATTATGAAC 5358 Jiang et al. 1998

APOBR AATCAGAGAGTTGGTCTGAAAAAT Jiang et al. 1998

DMP1 Den12(Ex-F) GATGAAGACGACAGTGGAGATGACACCTT 5155 Toyosawa et al. 1999

Den2(Ex-R) ATCTTGGCAATCATTGTCATC Toyosawa et al. 1999

Den2a(Ex-F) GACACCTTTGGTGATGA Van Den Bussche et al. 2003

Den10(Ex-R) GTTGCTCTCTTGTGATTTGCTGC Van Den Bussche et al. 2003

DenA(In-F) TGCARAGYGAYGATCCAGACAC Van Den Bussche et al. 2003

DenB(In-R) TGATTCTCTTGATTTGACACTGG Van Den Bussche et al. 2003

DenC(In-F) ACCTCCAGTCACTCAGAAG Van Den Bussche et al. 2003

DenD(In-R) GGATNGCTTTCWGAACTGRAGG Van Den Bussche et al. 2003

PRKC1 BatPKa(F) CTTGTCAATGATGATGAGG 4045 Eick et al. 2005

BatPKb(R) CCTATTTTAAAATATGAAAGAAATC Eick et al. 2005

RabbitPK(F) AAACAGATCGCATTTATGCAAT Matthee et al. 2004

RabbitPK(R) TGTCTGTACCCAGTCAATATC Matthee et al. 2004

RAG2 F1(Ex-F) GGCYGGCCCAARAGATCCTG 5361 Baker et al. 2000

F1B(Ex-F) ATCCTGCCCCACTGGAGTTTTC Baker et al. 2000

R2(Ex-R) GRAAGGATTTCTTGGGCAGGAGT Baker et al. 2000

F1int(In-F) GRACAGTCGAGGGAARAGCATGG Baker et al. 2000

F2(In-F) TTTGTTATTGTTGGTGGCTATCAG Baker et al. 2000

F2int(In-F) GGAYTCCACTCCCTTTGAAGA Baker et al. 2000

R1(In-R) AACYTGYTTATTGTCTCCTGGTATGC Baker et al. 2000

R1int(In-R) GGGGCAGGCASTCAGCTAC Baker et al. 2000

R2int(In-R) GCAGCAWGTAATCCAGTAGC Baker et al. 2000

Myotis179F(Ex-F) CAGTTTTCTCTAAGGAYTCCTGC 5254 Stadelmann et al. 2007

Myotis1458R(Ex-R) TTGCTATCTTCACATGCTCATTGC Stadelmann et al. 2007

Myotis428F(In-F) ATGTGGTATATAGTCGAGGGAAGAGC Stadelmann et al. 2007

Myotis968R(In-R) CCCATGTTGCTTCCAAACCATA Stadelmann et al. 2007

STAT5A BatSTATa(F) CTGCTCATCAACAAGCCCGA 4862 Eick et al. 2005

BatSTATb(R) GGCTTCCGGTTCCACAGGTTGC Eick et al. 2005

ArtiSTATa(F) GAAGAAACATCACAAGCCCC 5160 Matthee et al. 2004

ArtiSTATb(R) AGACCTCATCCTTGGGCC Matthee et al. 2004

THY BatTHYa(F) GGGTATGTAGTTCATCTTACTTC 4259 Eick et al. 2005

BatTHYb(R) GGCATCCTGGTATTTCTACAGTCTTG Eick et al. 2005

RabbitTHYa(F) CATCAACACCACCATCTGTGC 5259 Matthee et al. 2004

RabbitTHYb(R) CACTTGCCACACTTACAGCT Matthee et al. 2004


filogenetica molecular myotis

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