Fylogenetická Reprezentace Syndromu Bílého Nosu U Netopýrů Bakalářská Práce Matej Dolinay

Home , Glischropus, Laephotis, Murininae, Scotoecus

MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA ÚSTAV BOTANIKY A ZOOLOGIE

Fylogenetická reprezentace syndromu bílého nosu u netopýrů Bakalářská práce Matej Dolinay

Vedoucí práce: Mgr. Natália Martínková, Ph.D. Brno 2013

Bibliografický záznam

Autor: Matej Dolinay Přírodovědecká fakulta, Masarykova univerzita Ústav botaniky a zoologie Fylogenetická reprezentace syndromu bílého nosu u Název práce: netopýrů

Studijní program: Biologie

Studijní obor: Ekologická a evoluční biologie

Vedoucí práce: Mgr. Natália Martínková, Ph.D.

Akademický rok: 2012/2013

Počet stran: 60

Klíčová slova: Netopýři; Vespertilionidae; Syndrom bílého nosu; Fylogeneze; Geomykóza; Fylogenetická reprezentace

Bibliographic Entry

Author Matej Dolinay Faculty of Science, Masaryk University Department of Botany and Zoology

Title of Thesis: Phylogenetic representation of white-nose syndrome in bats Degree programme: Biology

Field of Study: Ecological and evolutionary biology

Supervisor: Mgr. Natália Martínková, Ph.D.

Academic Year: 2012/2013

Number of Pages: 60 Keywords: Bats; Vespertilionidae; White-nose syndrome; Phylogeny; Geomycosis; Phylogenetic representation

Abstrakt

Syndrom bílého nosu (WNS) je nedávno objevené infekční onemocnění hibernujících netopýrů, které způsobuje mikroskopická houba Geomyces destructans. V Severní Americe byl WNS diagnostikován u sedmi druhů a u několika z nich byla zaznamenána masová úmrtnost po propuknutí nemoci. V Evropě je potvrzených osm druhů infikovaných geomykózou a jeden byl diagnostikován s WNS. Masová úmrtnost nebyla v Evropě zaznamenána. Pro studium fylogenetických vztahů netopýrů ohrožených infekcí WNS jsem rekonstruoval fylogenezi 252 druhů z čeledí Vespertilionidae, Cistugidae a Miniopteridae pomocí spojeného datasetu složeného ze 13 mitochondriálních a jaderných lokusů metodou maximální věrohodnosti. Druhy netopýrů s diagnostikovaným WNS jsou polyfyletické a patří k fylogeneticky vzdáleným rodům Myotis, Eptesicus a Perimyotis. V rámci rodu Myotis se druhy s potvrzeným WNS a geomykózou nachází na různých pozicích tohoto kladu. Analýza fylogenetické reprezentace ukázala, že druhy postižené geomykózou jsou fylogeneticky klastrovány a druhy trpící WNS jsou signifikantně klastrovány, ale onemocnění může napadat i vzdáleně příbuzné druhy s podobnou ekologií. Ve výsledcích předkládám komplexní fylogenezi čeledi Vespertilionidae, která může poskytnout informace o druzích, které by mohly být postiženy syndromem bílého nosu v budoucnosti.

Abstract

The white-nose syndrome (WNS) is an emerging infectious disease of hibernating bats, which is caused by a microscopic fungus Geomyces destructans. In North America, seven species were diagnosed with WNS and several of them suffer mass mortality following the disease outbreak. In Europe, eight species are known to be infected with geomycosis and one was diagnosed with WNS. No mass mortality is associated with WNS in Europe. To study phylogenetic relationships of bat species prone to WNS, I reconstructed a phylogeny of 252 species of families Vespertilionidae, Cistugidae and Miniopteridae from a concatenated dataset including 13 mitochondrial and nuclear loci using the maximum likelihood aproach. The bat species affected with WNS are polyphyletic and they belong to distantly related genera Myotis, Eptesicus and Perimyotis. Within Myotis, species with confirmed WNS and geomycosis are found across the Myotis clade. The analysis of phylogenetic reperesentation confirmed that bats with geomycosis are phylogenetically clustered and those with WNS are significantly clustered on the phylogeny, but the disease might affect distantly related taxa with similar ecology. The results provide a comprehensive phylogeny of family Vespertilionidae, that may provide information about species pairs that could become affected with white-nose syndrome in the future.

Poděkování

Na tomto místě bych chtěl poděkovat Mgr. Natálii Martínkové, Ph.D., která vedla moji práci, poskytovala mi odpovědi na mé otázky a věnovala svůj čas tomu, aby mě naučila zásadám pro vytvoření kvalitní vědecké práce.

Prohlášení

Prohlašuji, že jsem svoji bakalářskou práci vypracoval samostatně s využitím informačních zdrojů, které jsou v práci citovány.

Brno 05. měsíce 2013 ……………………………… Jméno Příjmení

TABLE OF CONTENTS 1. Introduction…………………………………………………..…...…..9

1.1. Phylogenetics………………………………………………..……...9

1.2. Phylogeny of bats……………………………………………..…....10

1.3. White-nose syndrome in terms of bat ecology………………….12

1.4. Aims……………………………………….…………………………17

2. Materials and methods…………………………….…….….……...18

3. Results……………………………………..…………….……………22

4. Discussion……………………………..…………………….……….46

4.1. Phylogeny of vespertilionids………………………………………47

4. 2. The phylogenetic representation of white-nose syndrome in bats...... 50

5. References……………………………………………………………53

1. INTRODUCTION

Pathogenic organisms infect hosts depending on their ability to overcome their defence mechanisms, entering into an evolutionary arms race between the pathogen and the host. In co-evolution between hosts and pathogens, there is often a presence of host switching to closely related species to the one which was already affected (IRWIN et. al. et al. 2012). To study the preferences for non-related or closely related species, I investigated phylogenetic representation of a fungal infection in vespertilionid bats. For the first step in understanding the phylogenetic aspect of host switching , it is important to analyze the phylogeny of the particular group. In this case, the pathogen is a microscopic fungus and hosts are representatives of vespertilionid bats.

1.1. Phylogenetics

One of the most basic knowledge about evolution is that all living organisms have one common ancestor (FLEGR 2005). A scientific discipline called phylogenetics studies evolutionary relationships of organisms. Phylogenetic trees are used as the graphic depiction of reconstruction of the lineage from the last single ancestor to descendants (O’HARA 1998). According to GREGORY (2008), evolutionary tree ” is a diagrammatic depiction of biological entities that are connected through common descent, such as species or higher-level taxonomic groupings“. Trees are rooted if we know the common ancestor (or unrooted in case of no known ancestor), the branching points represent internal nodes and the parts between them are called internal branches. Taxa with position on the terminal branches are called terminal taxa. To suggest a relationship between taxa, we calculate the length of the tree, which means total number of steps to reach the terminal position (LIPSCOMB 1998). The main unit of phylogenetics is a clade, a monophyletic group, which consists of an ancestor and all its descendants (BAUM 2009). Clades are the taxonomic units used for simplification of systematics because there is an upcoming trend of using too many categories in nomenclature (HENNIG 1966). However, the traditional system is still widely followed and will be used also in

2 this study. It is based on classification to ranks, which together form the taxonomic hierarchy (genus, family, class).

1.2. Phylogeny of bats

Bats form a clade in group Laurasiatheria and despite the fact, that they are the second biggest group of mammals (20 % of mammalian species are bats, JONES et al. 2002), their evolutionary history is still quite a mystery. It is because of missing fossil material and enigmatic position of some lineages placed inside Chiroptera (TEELING et al. 2005). After revealing that bats are a monophyletic group (Pteropodidae was suggested to be closely related to Dermoptera or Primata before, HUTCHEON & GARLAND 2004), the phylogeny inside Chiroptera needed to be resolved. The division into two suborders, Megachiroptera (nonecholocating bats) and Microchiroptera (echolocating bats) was proved incorrect by molecular studies (TEELING et al. 2002, HUTCHEON & GARLAND 2004, VAN DEN BUSSCHE & HOOFER 2004). HUTCHEON & GARLAND (2004) showed that members of Megachiroptera and Microchiroptera are not actually divided by their body size, but there is a considerable overlap among them. Thus, new systematics of bats was proposed and comprised of Yinpterochiroptera (including families Craseonycteridae, Pteropodidae, Hipposideridae, Megadermatidae, Rhinolophidae, and Rhinopomatidae) and Yangochiroptera (Emballonuridae and Nycteridae with other former microbats) instead of Microchiroptera and Megachiroptera (TEELING et al. 2005). The names Pteropodiformes and Vespertilioniformes have also been proposed for these suborders (HUTCHEON & KIRSCH 2006). The paraphyly of Microchiroptera means, that echolocation has originated twice in bats, or only once and it was secondarily lost in Pteropodidae (EICK et al. 2005). This study will focus on Vespertilionidae. The high-level relationships inside this family are still not completely resolved, but it includes almost one-third of recent bat species (SIMMONS 2005). Multiple subfamilies were recognised

3 according to morphological characters in the past, specifically SIMMONS & GEISLER (1998) recognized Vespertilioninae, Murininae, Miniopterinae and Kerivoulinae. Miniopterinae was raised to a family status, because of evidence from molecular and morphological data in later studies (EICK et al. 2005, VAN DEN BUSSCHE & HOOFER 2004) and this was again confirmed by MILLER- BUTTERWORTH et al. (2007). Later, some changes were made in the classification of the family. Genus Myotis was previously included in Vespertilioninae, but SIMMONS (2005) has changed its classification into Myotinae with genera Cistugo and Lasionycteris. However, Cistugo was later reclassified to its own family Cistugidae according to the genetic distance from Vespertilionidae (LACK 2010). Lasionycteris was found to be inside Vespertilioninae by ROEHRS et al. (2010). Kerivoulinae was splitted into two subgenera Kerivoula and Phoniscus, Murininae into subgenera Murina, Harpiola (JONES et al. 2002) and Harpiocephalus (SIMMONS 2005). The last recent valid subfamily, Vespertilioninae, comprised of seven tribes – Antrozoini, Lasiurini, Nycticeiini, Nyctophilini, Plecotini, Scotophilini and Vespertilionini according to SIMMONS (2005). However, ROEHRS et al. 2010 validated support only for Antrozoini, Lasiurini, Scotophilini and Vespertilionini, and then suggested existence of two tribes – the perimyotine group and the hypsugine group (Nycticeiini, Eptesicini and Plecotini remained unresolved). This classification still remains valid (Fig. 1). SIMMONS & GEISLER (1998) have not classified Antrozous to Vespertilionidae (Antrozoidae), but mitochondrial DNA analyses proved its position inside Vespertilioninae (HOOFER & VAN DEN BUSSCHE 2003).

Fig. 1 Cladogram based on phylogenetic analysis of Vespertilionidae published in ROEHRS et al. (2010).

1.3. White-nose syndrome in terms of bat ecology White-nose syndrome is an infectious disease of hibernating bats (BLEHERT et al. 2009). It has been so far found only in species from family Vespertilionidae (Tab. 1), which belongs to Yangochiroptera. LORCH et al. (2011) proved, that the primary cause of white-nose syndrome was the fungus Geomyces destructans by showing that exposition of healthy little brown bats (Myotis lucifugus) to the pure culture of G. destructans has triggered the infection. The analysis of rDNA gene sequence has placed the microscopic fungus causing the white-nose syndrome in the genus Geomyces (Ascomycota, Leotiomycetes, Helotiales, Myxotrichaceae). It has well recognizable asymmetrically curved conidia different from any other member of the genus Geomyces (GARGAS et al. 2009). The very first evidence of this pathogen was

5 documented in Howe Cave in state New York on 16th February 2006 (BLEHERT et al. 2009). Geomyces destructans is adapted to cold and can be found on exposed skin of bat hibernacula, which have the optimal temperature for growth of the hyphae (5-10 °C). WNS causes severe interventions to the life of hibernating bats. Infected bats have abnormal behaviour, small fat reserves, white fungal areas on muzzle and ears, or damaged wing tissues diagnosed by a histopathological examination of the skin. The infection is transmitted by close contact among individuals in the colony (WILDER et al. 2011). More than million individuals have died annually with average mortality of 73 % since 2006, so this is one of the most dramatic declines of mammal population ever observed (CRYAN et al. 2010). The probable cause of massive mortality is the intrusion of normal cycle during torpor including arousals in hibernating bats. It is likely connected to the evaporative water loss, which influences the arousal frequency during hibernation and leads to dehydratation and death (WILLIS et al. 2011). The dehydration is therefore an important aspect to observe in species infected by geomycosis. The observations showed that mortality in bats is largely dependent on colony location and microclimate. Very large colonies in big caves or the ones in a humid enviroment have a hight level of early mortality (WILDER et al. 2011). This is true in North America. The WNS is currently detected in 7 species from North America, namely Perimyotis subflavus, Eptesicus fuscus, Myotis grisescens, M. leibii, M. lucifugus, M. septentrionalis and M. sodalis. The fungus G. destructans has been found also on M. austroriparius and M. velifer, but without deadly consequences. The endangered Corynorhinus townsendii Cooper, 1837 is found in the affected areas, but has not been confirmed to be infected yet (BLEHERT et al. 2009, METEYER et al. 2009, US FWS 2011). In Europe, the only species with detected WNS is M. myotis (PIKULA et al. 2012) and six additional species were diagnosed with geomycosis (MARTÍNKOVÁ et al. 2010, WIBBELT et al. 2010). WNS was found in representatives of two subfamilies of Vespertilionidae (Myotinae and Vespertilioninae) and in two (putative) tribes within

Vespertilioninae (Vespertilionini and perimyotine group) according to the latest classification (Tab. 1). During the 2012/ 2013 hibernating season the WNS was confirmed in 22 states of the USA and five Canadian provinces, namely Alabama, Connecticut, Georgia, Illinois, Delaware, Indiana, Kentucky, Maine, Maryland, Massachusetts, Missouri, New Hampshire, New Jersey, New York, North Carolina, Ohio, Pennsylvania, Tennessee, Vermont, Virginia, West Virginia, New Brunswick (Canada), Prince Edward Island (Canada), Nova Scotia (Canada), Ontario (Canada) and Quebec (Canada), and it was suspected in Iowa and Oklahoma (Fig. 2) [United States Geological Survey (USGS), National Wildlife Health Center 2013, available at http://www.nwhc.usgs.gov/disease_information/white-nose_syndrome/index.jsp (accessed March 21, 2013)]. The first genetically confirmed record from Europe is from Germany in 2008 (WIBBELT et al. 2010). Three years after the first discovery in North America, this fungus was found in France (PUECHMAILLE et al. 2010) and the Czech Republic (MARTÍNKOVÁ et al. 2010) and very soon it became clear, that it actually has pan-European distribution (PUECHMAILLE et al. 2011a). Geomyces destructans was found to be widespread in the Czech Republic and Slovakia (MARTÍNKOVÁ et al. 2010). The presence of WNS in Europe was showed by histopathology on Myotis myotis by PIKULA et al. (2012). In Europe, no mass mortality of infected bats occured, although there were pathological changes confirmed on their skin (MARTÍNKOVÁ et al. 2010, PUECHMAILLE et al. 2011b, PIKULA et al. 2012). WARNECKE et al. (2012) suggested that Geomyces destructans has only recently arrived to North America as an invader from Europe. The other possibility is, that the fungus is native to both continents and has become more pathogenic because of environmental speciation or mutation in North America, or as PUECHMAILLE et al. (2011b) speculated, it could be transported over great distances by bats (colonisation of Europe by Myotis blythii from Asia).

Fig. 2 Geographic distribution of white-nose syndrome in North America on the 21th of March 2013 depicted by United States Geological Survey (USGS), National Wildlife Health Center 2013 (available at http://www.nwhc.usgs.gov/disease_information/white- nose_syndrome/index.jsp).

Tab. 1 Classification of vespertilionid groups with G. destructans and white- nose syndrome (Myotinae and Vespertilioninae) according to SIMMONS (2005) and ROEHRS et al. (2010).

SIMMONS (2005) ROEHRS et al. (2010) Vespertilioninae Myotinae Antrozoinae Vespertilioninae Myotinae Missing data Plecotini Lasionycteris Antrozous Plecotini Myotis Nyctophilini Barbastella Cistugo Bauerus Barbastella Nyctophilus Corynorhinus Myotis Corynorhinus Pharotis Euderma Euderma other genera: Idionycteris Idionycteris Philetor Otonycteris Otonycteris Ia Plecotus Plecotus Mimetillus Lasiurini Lasiurini Histiotus Lasiurus Lasiurus Falsistrellus Nycticeiini Nycticeiini/ Eptesicini Eudiscopus Rhogeessa Arielulus Scotozous Baeodon Lasionycteris Glischropus Nycticeinops Eptesicus Scoteinus Nycticeius Glauconycteris Scoteanax Scoteanax Nycticeius Hesperoptenus Scotoecus Scotomanes Scotorepens Scotomanes Antrozoini Scoteinus Antrozous Scotophilus Rhogeessa Scotorepens Baeodon Eptesicini Bauerus Eptesicus Scotophilini Arielulus Scotophilus Hesperoptenus Perimyotine group Pipistrellini Perimyotis Glischropus Parastrellus Nyctalus Hypsugine group Pipistrellus Chalinolobus Perimyotis Hypsugo Parastrellus Laephotis Scotozous Neoromicia Vespetilionini Nycticeinops Chalinolobus Tylonycteris Eudiscopus Vespadelus Falsistrellus Vespertilionini Glauconycteris Nyctalus Histiotus Pipistrellus Hypsugo Scotoecus Ia Vespertilio Laephotis Mimetillus Neoromicia Philetor Tylonycteris Vespadelus Vespertilio Nyctophilini Nyctophilus Pharotis

Fig. 3 Distribution of confirmed and suspected records of G. destructans on hibernating bats in Europe according to PUECHMAILLE et al. (2011). Genetically confirmed records of the pathogen are shown in red (circles - PUECHMAILLE et al. 2011, triangles -published records), photographic evidence in yellow, visual reports in green colour. Black dots represent the dead bats from Northern France without genetic analysis proving the presence of Geomyces destructans (available at DOI: 10.1371/journal.pone.0019167).

1.4. Aims The main aim to achieve in my work was to reconstruct a comprehensive phylogeny of family Vespertilionidae and to investigate phylogenetic relatedness of infected species. The analysis of phylogenetic representation of the WNS in bats should give a comprehensive conclusion about the dispersal of this trait in the phylogeny. Based on this information, I will hypothesize about species pairs that could become affected with the WNS in the future.

2. MATERIALS AND METHODS

My analysis included 13 mitochondrial and nuclear loci (Tab. 2), which comprised a dataset including families Vespertilionidae, Cistugidae and Miniopteridae with 252 species. The sequences were obtained from the GenBank database (BENSON et al. 2010) with open-access to all published DNA sequences. Geneious software 5.6.2 (KEARSE et al., 2012) was used for downloading, ordering and aligning sequences. A substitution model was chosen for gene trees by ModelGenerator software (KEANE et al. 2006). It uses the Akaike Information Criterion (AIC), the Bayesian Information Criterion (BIC) and hierarchical likelihood-ratio test (hLRT) to select the best-fitting nucleotide substitution models from alignments in Fasta or Phylip format. The Bayesian Information Criterion (BIC) was used to select the appropriate model. My study also uses a concatenated dataset, created in SequenceMatrix 1.7.8 software (VAIDYA et al. 2011). Genes were concatenated by dragging and dropping NEXUS files with aligned sequences into the program window. Then the alignments of every gene were connected together to form a concatenated dataset. Missing data were replaced by question mark, gaps by dash. Partitions for the concatenated dataset were optimalized in PartitionFinder software (LANFEAR et al. 2012) (Tab. 3). It allows millions of partitioning schemes to be compared in realistic timeframes and finds the objective selection of partitioning schemes for large multi-locus DNA datasets. Gene trees were analysed by Bayesian method in MrBayes 3.2. (RONQUIST et al. 2012). The Bayesian inference uses Markov chain Monte Carlo (MCMC) algorithms to estimate the posterior distribution of model parameters for the observed data. It is a consequence of prior probability and likelihood. In the gene trees, MCMC ran for 4 million generations, sampled every 1000th (resulting in 4000 samples from the posterior probability distribution), and run convergencediagnostics were calculated every 5000 generations. Rag2 and Coi datasets were calculated with 5 million generations. The temperature was set to 0.09 (for the Prkc1 dataset to 0.07 and the Rag2 dataset to 0.08). Six chains and two runs were used. One thousand samples from the beginning of the

11 chain were discarded (burnin=1000). Miniopteridae and Cistugidae were used as outgroups.

Tab. 2 Location of loci, number of species, alignment length and substitution models used for gene trees.

Gene Location Number of species Length Substitution model 12S mitochondrial 118 1160 bp HKY +I Coi mitochondrial 102 658 bp HKY +I Cytb mitochondrial 138 1140 bp GTR +I Nd1 mitochondrial 74 957 bp HKY +I Nd2 mitochondrial 21 1044 bp GTR +I Apob nuclear 110 282 bp HKY + Γ Dmp1 nuclear 109 1032 bp HKY + Γ Prkc1 nuclear 117 445 bp GTR + Γ Rag2 nuclear 156 1244 bp GTR + Γ Stat5a exon nuclear 37 485 bp HKY + Γ Stat5a intron nuclear 64 514 bp HKY + Γ Thy exon nuclear 46 567 bp HKY + Γ Thy intron nuclear 65 563 bp HKY + Γ

The concatenated dataset was analysed in RAxML version 7.2.6 software (STAMATAKIS 2006). It uses a maximum likelihood (ML) method to reconstruct the phylogeny and calculate the bootstrap support for nodes. When given a dataset and a substitution model, this method estimates the phylogeny and the model's parameters. RAxML is very efficient in analysing large phylogenies with the best ML scores and a smaller amount of time compared to other ML methods (LIU et al. 2011). Likelihood of the final tree was evaluated and optimized under GTR + Γ substitution model for each partition (Tab. 3). Model parameters were estimated up to an accuracy of 0.1 log-likelihood units.

Tab. 3 Partitions and substitution model used according to the PartitionFinder analysis.

Partition number Genes Substitution model

0 12S GTR + Γ 1 Coi GTR + Γ 2 Cytb, Nd1, Nd2 GTR + Γ 3 Apob, Rag2 GTR + Γ 4 Dmp1 GTR + Γ 5 Prkc1 GTR + Γ 6 Stat5a intron, exon GTR + Γ 7 Thy intron, exon GTR + Γ

The phylogenetic representation of the G. destructans infection and the white- nose syndrome from the concatenated dataset was done in Phylocom 4. 2 software (WEBB et al. 2008). It measures a phylogenetic signal, trait similarity and correlated evolution in phylogenetic community structure through calculation of numerous metrics. The model comstruct was used, which analyses the phylogenetic structure with two metrices: mean phylogenetic distance (MPD) and mean nearest taxon distance (MNTD). MPD calculates the average distance among two random taxa chosen from the sample, MNTD the average distance to the closest relative for each taxon in the sample. Maximum likelihood tree was first pruned (with the sampleprune function) to include groups of species according to their presence in temperate zones where bats hibernate, which is crucial for the existence of G. destructans. The tree with hibernating bats from North America and Europe was then used as an input file to the Phylocom analysis, table with presence of G. destructans and WNS was used as a sample (Tab. 4).

Tab. 4. Presence of geomycosis and white-nose syndrome in bats from North America and Europe.

Geomycosis Reference White-nose syndrome Reference

North America

BLEHERT et al., Eptesicus fuscus BLEHERT et al. 2009 Eptesicus fuscus 2009

Myotis grisescens US FWS 2011 Myotis grisescens US FWS 2012

Myotis leibii US FWS 2011 Myotis leibii US FWS 2011

BLEHERT et al., Myotis lucifugus BLEHERT et al. 2009 Myotis lucifugus 2009 BLEHERT et al., Myotis septentrionalis BLEHERT et al. 2009 Myotis septentrionalis 2009 METEYER et al., Myotis sodalis METEYER et al. 2009 Myotis sodalis 2009 BLEHERT et al., Perimyotis subflavus BLEHERT et al. 2009 Perimyotis subflavus 2009

Myotis austroriparius US FWS 2011

Myotis velifer US FWS 2011

Europe

Myotis myotis WIBBELT et al. 2010 Myotis myotis PIKULA et al., 2012 Myotis blythii WIBBELT et al., 2010

Myotis brandtii WIBBELT et al., 2010

Myotis dasycneme WIBBELT et al., 2010

Myotis daubentonii WIBBELT et al., 2010

MARTÍNKOVÁ et al., Myotis mystacinus 2010 MARTÍNKOVÁ et al., Myotis nattereri 2010

3. RESULTS

Thirteen gene trees and one tree from the concatenated dataset were the output of the phylogenetic analyses. The length of the alignments varied from 118 to 1244 base pairs (Tab. 2). Proportion of gaps and completely undetermined characters in this alignment was 64.51%. HKY (HASEGAWA 1985) and GTR (TAVARE 1986) substitution models were used in the Bayesian analyses of gene trees according to the result of ModelGenerator. In the maximum likelihood (ML) phylogeny of the conctenated dataset, GTR substitution model was used for all partitions. Each tree will be described in detail. In the 12S rRNA vespertilionid tree, genus Scotophilus was a sister taxon to all other taxa from Vespertilionidae. In the strongly supported lineage of other species, there was a strong support for subfamilies Kerivoulinae, Murininae and Myotinae. Vespertilioninae was paraphyletic. Apart from Scotophilini, which branched at the base of the group, strongly supported tribes from Vespertilionae were Lasiurini and Antrozoini. Another strongly supported cluster of species included representatives of Nycticeiini and the hypsugine group (Laephotis, Neoromicia, Eptesicus, Hypsugo, Nyctophilus, Chalinolobus). Other strongly supported branches were monophyly of Plecotus and a lineage including Eptesicus dimissus and Tylonycteris (Fig. 4). The position of Scotophilus in the Coi tree was the same as in the previous tree. There was not a strong support for the whole clade of all other taxa. Within it, the strongly supported groups were subfamilies Kerivoulinae, Murininae and tribes Antrozoini, Lasiurini and part of Nycticeiini including Eptesicus, Glauconycteris, Scotomanes, Lasionycteris and Nycticeius (Fig. 5). In the tree from Cytb, Scotophilus was a sister taxon to the lineage consisting of representatives of all Vespertilionidae subfamilies with strongly supported Kerivoulinae and Murininae. There was strong support for a branch consisting of representatives of tribes Vespertilionini and Nycticeiini. One lineage included Eptesicus, Laephotis, Tylonycteris and Vespertilio, the second one Pipistrellus and Nyctalus (Fig. 6).

The Nd1 tree consisted of two strongly supported lineages except the outgroup (Miniopteridae). The first one included strongly supported branches of Myotinae and Murininae, the second a cluster of species from several tribes within Vespertilioninae (Nycticeiini, Plecotini and Vespertilionini with Hypsugo ariel and H. savii). Within Vespertilionini, there were two strongly supported lineages, Pipistrellus lineage and Vespertilio lineage (Fig. 7). In the Nd2 tree, there was a strong support for the Myotinae lineage, which was sister to all other taxa in the tree except the outgroup. Pipistrellus javanicus and Tylonycteris pachypus formed a group together with Antrozous pallidus in the cluster that was a sister group to the representatives of Kerivoulinae. This group as a whole was not strongly supported (Fig. 8). The Apob tree had only a few strongly supported nodes. The outgroup (Miniopteridae and Cistugidae) included also some taxa from Vespertilionidae (Neoromicia rendalli, Nyctalus noctula, Eptesicus furinalis, Rhogeessa alleni, Myotis yumanensis). Lineage of Scotophilus was sister to all other taxa from Vespertilionidae, and the branching of most other groups was unclear in this tree (Fig. 9). The tree resulting from the Dmp1 sequences included strongly supported group representatives of Vespertilionidae. The first lineage consisted of species from subfamilies Kerivoulinae, Myotinae and Murininae. In the second lineage, Scotophilini was sister to all other taxa. The strong support was for tribes Antrozoini and Lasiurini (Fig. 10). The outgroup of Prkc1 tree was not strongly supported, Kerivoula pellucida was included into the species from Miniopteridae and Cistugidae. Most branches within the big cluster of species from Vespertilionidae were not resolved. Myotis pilosus and M. adversus form a group with a very long branch, which can indicate incorrect sequences or accelerated mutation rate on the branch (Fig. 11). The Rag2 tree included strongly supported and well resolved Murininae, Kerivoulinae and Myotinae. Among Vespertilioninae, strongly supported tribes were Scotophilini, Lasiurini and Antrozoini. There was strong support for the group consisting of taxa from hypsugine group and Vespertilionini (Fig. 12).

There were three taxa from Vespertilionidae in the outgroup of the tree from exon sequences of Stat5a exon – Laephotis namibensis and Rhogeessa alleni. Myotis tricolor was sister taxon to all other species within the second cluster in the tree. There was strong support for Lasiurini (only Lasiurus xanthinus was not included in the group) and Nycticeiini with L. xanthinus. The last supported group was represented by genera from Antrozoini, hypsugine group and Nycticeiini (Fig. 13). In the Stat5a intron tree, unsupported Myotinae formed a sister group to a well-supported lineage with the subfamilies Murininae and Kerivoulinae. This locus showed supported Vespertilioninae, with clear tribes Antrozoini and Scotophilini. There were two supported lineages of representatives of the tribus Nycticeiini (three species of Eptesicus and Nycticeius humeralis and branch with one species from genus Eptesicus, Chalinolobus and Neoromicia). Species from Plecotini (Corynorhinus and Plecotus branch) were located on two strongly supported branches on different lineages on the tree (Fig. 14). In the tree from the Thy exon sequences, Myotis tricolor was a sister taxon to all other included representatives from Vespertilionidae. Strongly supported groups were Lasiurini, Vespertilionini, a group formed by sequences of Histiotus macrotus and genera Eptesicus and Glauconycteris. Another strongly supported branches were those of Scotomanes ornatus and Tylonycteris pachypus, Hypsugo eisentrauti and Nycticeinops schleiffeni, Eptesicus vulturnus and Hypsugo savii and Rhogeessa alleni and Scotophilus dinganii. There is also a strong support for the branch of Chalinolobus gouldii and Histiotus magellanicus (Fig. 15). The last gene tree represented the analysis of the Thy intron sequences. Strongly supported subfamilies were Murininae and Kerivoulinae. Within Vespertilioninae, there was strong support only for tribes Scotophilini and Antrozoini. Rhogeessa parvula had a very long branch, which may indicate an incorrectly annotated sequence or accelerated mutation rate (Fig. 16). Eight distinct data partitions with joint branch length optimization were used for the ML phylogeny reconstruction based on the concatenated alignment (Tab. 3). All partitions were analysed with the GTR substitution matrix and rate

17 heterogeneity modeled with the Γ distribution. The total alignment length was 10091 base pairs. Families Cistugidae and Miniopteridae were sister to Vespertilionidae (Fig. 17). Vespertilionidae included four subfamilies – Kerivoulinae, Murininae (Fig. 19), Myotinae (Fig. 20) and Vespertilioninae (Fig. 21). In two of them (Myotinae and Vespertilioninae), there was evidence of very rapid diversification. These lineages had very short branches at the basal positions and polytomies were present. Within Vespertilioninae, there was strong support for monophyly of tribes Scotophilini, Lasiurini, hypsugine group and perimyotine group (Fig. 18). Tribus Antrozoini was strongly supported with exclusion of Rhogeessa alleni and diverged as a basal group of Vespertilioninae. Tribus Plecotini was not significantly supported. There was strong support for two lineages in Nycticeiini, but the support was weak for the whole tribus. In Vespertilionini, genera Vespertilio, Tylonycteris and the species Eptesicus dimissus formed a distinct lineage from the supported Pipistrellus lineage and the overal support was also weak.

Fig. 4 12S rRNA gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 5. Coi gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 6 Cytb gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 7 Nd1 gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 8 Nd2 gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 9 Apob gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 10 Dpm1 gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 11 Prkc1 gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 12 Rag2 gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 13 Stat5a exon gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 14 Stat5a intron gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 15 Thy exon gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 16 Thy intron gene tree of vespertilionid bats from the Bayesian analysis, node labels show bayesian posterior probability

Fig. 17 Tree from concatenated dataset analysed in RAxML software, depictions of outgroup families (Cistugidae, Miniopteridae), subfamilies of Vespertilionidae (Kerivoulinae, Murininae, Myotinae and Vespertilioninae)

Fig. 18 Tree from concatenated dataset analysed in RAxML software, depictions of tribes within Vespertilioninae (tribes without strong support are marked with ?)

Fig. 19 The partial ML phylogeny from the concatenated dataset as displayed in Fig. 17, subfamilies Kerivoulinae and Murininae

Fig. 20 The partial ML phylogeny from the concatenated dataset as displayed in Fig. 17, subfamily Myotinae

Fig. 21 Fig. 20 The partial ML phylogeny from the concatenated dataset as displayed in Fig. 17, subfamily Vespertilioninae

The phylogenetic representation of the presence of G. destructans and white- nose syndrome was analyzed on the tree that included only hibernating bats from Europe and North America (Tab. 5). For G. destructans, the mean nearest taxon distance was significantly lower than expected by chance and taxa with trait were more closely related to sister taxa with trait than explained by chance (NTI > 0). The mean phylogenetic distance was significantly lower than expected by chance (p = 0.0), and the trait was phylogenetically clustered (NRI >0). For white-nose syndrome, the mean phylogenetic distance was significantly lower than expected by chance (p = 0.009), trait was phylogenetically clustered (NRI > 0). The mean nearest taxon distance was not significantly lower than expected by chance (p = 0.062). Here, taxa with white- nose syndrome were not more closely related to sister taxa with this trait than explained by chance.

Tab. 5 Analysis of phylogenetic representation with randomization method 0 (this null model shuffles species labels across the entire phylogeny and randomizes phylogenetic relationships among species). Ntaxa column shows number of species, MPD the mean phylogenetic distance, NRI the net relatedness index, MNTD the mean nearest phylogenetic taxon distance, MPD/MNTD .rnd value obtained from the null communities, .sd standard deviation from the null communities, .rankLow the number of null communities with MPD/MNTD values less than or equal to observed, .rankHi the number of null communities with MPD/MNTD values greater than or equal to observed, runs the number of runs to randomize over.

Values Infection GD WNS ntaxa 18 8 MPD 0.1626 0.1792 MPD.md 0.2709 0.2693 MPD.sd 0.0218 0.0369 NRI 4.9588 2.4415 MPD.rankLow 999 990 MPD.ranHi 0 9 MNTD 0.0927 0.1193 MNTD.rnd 0.1420 0.1732 MNTD.sd 0.0203 0.0350 NTI 2.4307 1.5419 MNTD.rankLow 993 937 MNTD.ranHi 6 62 runs 999 999

4. DISCUSSION The maximum likelihood phylogeny resolved multiple enigmatic relationships between tribes, genera and at the species level. The analysis of phylogenetic representation of white-nose syndrome provided comprehensive data about dispersal of this disease across the phylogeny of vespertilionid bats.

4. 1. Phylogeny of vespertilionids

Because of rapid radiation of major lineages within Vespertilionidae, the inference of evolutionary relationships for representatives of this family has been problematic. The most recent hypotheses were tested using phylogenetic methods, however, they were still unable to resolve many of the enigmatic positions in the tree (VOLLETH & HELLER 1994, VAN DEN BUSSCHE & HOOFER 2004, MAYER et al. 2007). The results of my analyses enabled me to evaluate the phylogeny of bats from family Vespertilionidae, and the 13 gene trees from mitochondrial and nuclear loci generated by the Bayesian method produced comparable results to previous analyses. In Apob, Prkc1 and Stat5a exon tree there was an evidence of incomplete lineage sorting, because some taxa were included in the evolutionary distant outgroup. The length of alignment for these genes was too short, and it probably contained too little variability to detect the phylogenetic signal. It was a case of Neoromicia rendalli, Nyctalus noctula, Eptesicus furinalis, Rhogeesa alleni and Myotis yumanensis in the Apob tree, and Laephotis namibensis and Rhogeessa alleni in the Stat5a tree and Kerivoula pellucida in the Prkc1 tree. Genes with rapid rates of evolution may be excluded in the future studies, because they can influence the results of phylogenetic analyses and the evolutionary signal is therefore suppressed (PHILIPPE & TELFORD 2006). It can be useful for mitochondrial loci. Families Cistugidae and Miniopteridae were sister to Vespertilionidae, which is consistent with the analyses published in previous studies (VAN DEN BUSSCHE & HOOFER 2004, EICK et al. 2005, MILLER-BUTTERWORTH et al.2007, LACK 2010). Vespertilionidae included four subfamilies – Kerivoulinae, Murininae, Myotinae and Vespertilioninae. According to affinities of Myotis to Kerivoulinae and Murininae, it was elevated to subfamily rank Myotinae

(VOLLETH & HELLER 1994, LACK et al. 2010) and my analysis is consistent with this conclusion, because it was a sister subfamily of these two on the maximum likelihood tree. From traditionally recognized tribes within Vespertilioninae, the maximum likelihood based on the concatenated supermatrix method in this study provided support for Antrozoini (without Rhogeesa alleni), Lasiurini, Scotophilini, the hypsugine group and the perimyotine group. The last complex analysis of ROEHRS et al. (2010) provided phylogenetic information for 8 out of the 10 tribes previously proposed in various classifications of Vespertilioninae (Antrozoini, Eptesicini, Lasiurini, Myotini, Nycticeiini, Nyctophilini, Pipistrellini, Plecotini, Scotophilini, and Vespertilionini) and suggested the existence of two new detected lineages – perimyotine group and hypsugine group. My study provided support for validity of these tribes and clarifies the phylogenetic relationships between other supported lineages within Vespertilioninae. Other recognized tribes (but without strong support) were Plecotini, Vespertilionini and Nycticeiini. SIMMONS (2005) included Scotophilus in Nycticeiini, but molecular analysis of HOOFER & VAN DEN BUSSCHE (2003) supported the existence of its own tribus. In my analysis, Scotophilini was strongly supported and well resolved. The classification of Lasiurus into separate tribus was recognized since it was described and it is consistent (HOOFER & VAN DEN BUSSCHE 2003). My maximum likelihood phylogeny also supports the monophyly of this genus. As in the study of HOOFER & VAN DEN BUSSCHE (2003), my tree from the concatenated dataset supported monophyly of the tribus Antrozoini with genera Antrozous and Rhogeessa. However, the support was strong only with exclusion of Rhogeessa alleni, which formed a sister branch to the other members of the tribe. The next supported group consisted of two species from the New World – Parastrellus hesperus and Perimyotis subflavus. The clade was named by ROEHRS et al. (2010) as perimyotine group, but their relationship was recognized already by HOOFER & VAN DEN BUSSCHE (2003). The tribe named hypsugine group by ROEHRS et al. (2010) consisted of genera Chalinolobus, Hypsugo, Laephotis, Neoromicia, Nycticeinops and Vespadelus (here named in older classification to Eptesicus). It was divided into

40 three lineages, whose relationship to each other is unresolved. One consisted of genera from Australia (Chalinolobus, Nyctophilus and species from genus Eptesicus currently classified as Vespadelus) with Hypsugo ariel and H. savii basal to this lineage. Another lineage included Nycticeinops, Neoromicia and Hypsugo eisentrauti, the third one Laephotis, Neoromicia capensis (here as Eptesicus capensis) and Hypsugo cadornae. Hypsugo eisentrauti formed a group with Nycticeinops, which supports a recommendation of HOOFER & VAN DEN BUSSCHE (2003) about its transfer to the genus Nycticeinops. These results suggest, that Hypsugo is probably paraphyletic and will require further investigation. The sequence of Eptesicus nasutus was sister to this tribe, but it had a very long branch, which can indicate an incorrectly identificated voucher. Other tribes were not supported as whole. Tribus Nycticeiini is problematic and only two lineages within it were strongly supported in my analysis. As ROEHRS et al. (2010) claimed, ”it is apparent that Arielulus, Eptesicus (including Histiotus), Glauconycteris, Lasionycteris, and Scotomanes form a tribal level clade, but more effort will be required to resolve the true position of Nycticeius and will have an impact on the nomenclature of this clade.“ However, the first strongly supported lineage in my analysis included Histiotus, Eptesicus, Scotomanes and Ia, the second one Arielulus, Glauconycteris, Lasionycteris and Nycticeius. According to these data, molecular evidence fails to support or reject the previously proposed recognition of tribe Nycticeiini (VOLLETH & HELLER 1994, HOOFER & VAN DEN BUSSCHE 2003, ROEHRS et al. 2010). The next tribus was composed of genera Nyctalus, Pipistrellus, Scotoecus, Tylonycteris, Vespertilio and Glischropus. There were three strongly supported lineages within this tribe. The first one included Pipistrellus, Nyctalus, Glischropus and Scotoecus, the second one was theVespertilio lineage and the third consisted of Tylonycteris and Eptesicus dimissus. Eptesicus dimissus included in this tribus needs a special comment, because it was found in a supported Tylonycteris clade. According to ROEHRS et al. (2010), ”1) this specimen represents a misidentifed Tylonycteris or 2) E. dimissus requires a position change to the genus Tylonycteris.“ The sequence in GenBank was collected from Laos, but type specimens of

Eptesicus dimissus come from Nepal and Thailand (SIMMONS 2005). The incorrect identification of the voucher is probably a reason, because morphology of this species supports the classification into the genus Eptesicus (MYERS et al. 2000). The last unsupported tribe is Plecotini. The validity of this tribe was tested only recently (HOOFER & VAN DEN BUSSCHE 2003, ROEHRS et al. 2010) and these studies were unable to support or reject the monophyly of Plecotini. The results of my analysis support more the rejection of validity of this tribe. Genera traditionally recognized in the tribe are Barbastella, Corynorhinus, Euderma, Idionycteris and Plecotus, later also Otonycteris was included (SIMMONS 2005). In my analysis, there were strongly supported lineages of Euderma with Idionycteris, Plecotus lineage, Corynorhinus lineage, Barbastella lineage and also Otonycteris lineage, but their relationships were poorly resolved. The position of Eudiscopus was unresolved in the tree. It formed a sister branch to Kerivoulinae and Murininae, indicating, but not proving, a relationship. There is only a little evidence about classification of this genus, in SIMMONS (2005) it was included in Vespertilionini. Three species with unclear status were included in my analysis. The erroneous reporting of the type locality (Uruguay instead of China) led to the dual naming of Myotis pilosus (Peters 1869). The second name was M. ricketti (Thomas, 1894) and according to SMITH & XIE (2008) M. pilosus has the priority. Maximum likelihood tree showed, that the sequences are almost identical, so the sequence of M. ricketti represents in fact M. pilosus. According to SIMMONS (2005), Myotis oxygnathus and Myotis blythii are separate species and sequences of these two were not closely related in the tree. My analysis provides additional support for this classification. The last one is Plecotus alpinus, which was proved to be a synonym of Plecotus macrobullaris by SPITZENBERGER et al. (2003). Two sequences included in my analysis are not identical and they were not sister taxa, so this result supports the validity of both species from the Plecotus lineage. Results presented here provide a robust phylogeny of Vespertilionidae. My study evaluates the general pattern in evolution of vespertilionid bats and

42 reconstructs a robust phylogeny with good resolution in deep nodes. Multiple enigmatic relationships between tribes, genera and at the species level were analysed and it is a well resolved source for future studies of evolutionary relationships of Vespertilionidae. More sequences and more taxa will be necessary to be included in the analysis to resolve the relationships of vespertilionid bats of particular lineages.

4. 2. The phylogenetic representation of white-nose syndrome in bats

Geomyces destructans and its accompanying disease white-nose syndrome are present only in hibernating bats. It is caused by the temperature preferences of the fungus (CRYAN et al. 2010), so only bat species from temperate zones were included in the analysis of phylogenetic representation. My results showed, that species with G. destructans were phylogenetically closely related to other species with this trait. This was mainly caused by the fact, that most of the species belong to a single genus Myotis. For white-nose syndrome, the species were also clustered on the phylogeny, but the nearest relatives were not significantly more often infected than expected by chance.Therefore, the trait representing presence of G. destructans was clustered on the tree mainly in subfamily Myotinae (G. destructans is present in other two species of Myotis without white-nose syndrome). Moreover, the only one bat species with confirmed white-nose syndrome in Europe is Myotis myotis (PIKULA et al. 2012). Taxa with white-nose syndrome were not more closely related to sister taxa with this trait than explained by chance, however, the histhopathological analysis for more species can include new data with potential to change the result. This analysis indicates, that this fungal pathogen prefers the myotine lineage of vespertilionids. However, the presence of white-nose syndrome in Eptesicus fuscus and Perimyotis subflavus shows, that the phylogenetic preference of it is probably not very strict. The phylogenetic aspect of white-nose syndrome could be matched closely together with ecological aspect, because closely related species can be similar in their ecology. WILDER et al. (2011) showed, that the larger colonies in big caves and colonies

43 in a more humid environment are easier for G. destructans to spread between individuals and the level of mortality is very hight here. LANGWIG et al. (2012) claimed, that ”in hibernating bats infected with Geomyces destructans, that impacts of disease on solitary species were lower in smaller populations, whereas in socially gregarious species declines were equally severe in populations spanning four orders of magnitude. However, as these gregarious species declined, we observed decreases in social group size that reduced the likelihood of extinction. In addition, disease impacts in these species increased with humidity and temperature such that the coldest and driest roosts provided initial refuge from disease.“ The role of the microclimate in the caves where bats hibernate and the size of their colonies is important for the speed of spreading of G. destructans and therefore it is much easier to detect the declines caused by this pathogen in the more socially living species. However, results of my analysis of phylogenetic representation showed, that geomycosis may be present in more species with smaller impact on their populations. Hibernating bats from North America overally use much bigger cave systems that those from Europe and the spreading of fungus is much quicker in such communities (WILDER et al. 2011). Moreover, available data suggest, that bats in Europe might have been historically exposed to G. destructans and have evolved a mechanism for resistance to this pathogen. This could be an explanation of the fact, that species from Europe do not suffer from mass mortality of their populations caused by presence of G. destructans (PUECHMAILLE et al. 2011). The results of my analysis of phylogenetic representation may provide information about species pairs that could become affected with white-nose syndrome in the future. The clustering of trait (presence of G. destructans) in the tree indicates, that other hibernating species from genera Myotis and Eptesicus are endangered from becoming infected by this pathogen. There is also a possibility, that species from other genera with similar ecology will suffer from white-nose syndrome in the future – Plecotus, Barbastella, Corynorhinus. Living in bigger colonies and missing evolutionary adaptation to survive the presence of white-nose syndrome might make North American species more

44 vulnerable to exposion of G. destructans, so the conservation priority is to prevent the further spreading to new areas and species. This study provides the evolutionary basis for white-nose syndrome in bats and similar concept could be also used to study other infectious diseases causing mass mortality in vertebrates.

REFERENCES

BAUM D. A. 2009: Species as Ranked Taxa, Society of Systematic Biologists, 58(1):74-86.

BENSON D. A., KARSCH-MIZRACHI I., LIPMAN D. J., OSTELL J. & SAYERS E.W. 2010: GenBank. Nucleic Acids Research 38 (Database issue), D46– D51. doi: 10.1093/nar/gkp1024.

BLEHERT D. S., HICKS A. C., BEHR M., METEYER C. U., BERLOWSKI-ZIER B.M., BUCKLES E.L., COLEMAN J. T. H., DARLING S. R., GARGAS A., NIVER R., OKONIEWSKI J. C., RUDD R. J. & STONE W. B. 2009: Bat White- Nose Syndrome: An Emerging Fungal Pathogen?, Science, 323: 227.

CRYAN P.M., METEYER C.U., BOYLES J.G. & BLEHERT D.S. 2010: Wing pathology of white-nose syndrome in bats suggests life-threatening disruption of physiology, BMC Biology, 8: 135.

DE QUEIROZ A., GATESY J. 2007: The supermatrix approach to systematics, Trends in Ecology and Evolution, 22(1): 34-41.

EICK G. T., JACOBS D.S. & MATTHEE C. A. 2005: A Nuclear DNA Phylogenetic Perspective on the Evolution of Echolocation and Historical Biogeography of Extant Bats (Chiroptera), Molecular Biology and Evolution , 22(9):1869–1886.

FLEGR J. 2005: Evoluční biologie, Academia, Praha.

GADAGKAR S. R., ROSENBERG M. S. & KUMAR S. 2005: Inferring Species Phylogenies From Multiple Genes: Concatenated Sequence Tree Versus Consensus Gene Tree, Journal of Experimental Biology (MOL DEV EVOL), 304B: 64–74.

GARGAS A., TREST M. T., CHRISTENSEN M., VOLK T. J. & BLEHERT D. S. 2009: Geomyces destructans sp. nov. associated with bat white-nose syndrome, Mycotaxon, 108: 147–154.

HASEGAWA M., KISHINO H., YANO T 1985: Dating of the human-ape splitting by a molecular clock of mitochondrial DNA, Journal of Moleculal Evolution, 22 (2): 160–74.

HENNIG W. 1966: Phylogenetic systematics, Urbana, University of Illinois Press.

HOOFER S. R. & VAN DEN BUSSCHE, R. A. 2003: Molecular phylogenetics of the chiropteran family Vespertilionidae, Acta Chiropterologica 5 (supplement): 1–63.

HUTCHEON J. M. & GARLAND T. 2004: Are Megabats Big?, Journal of Mammalian Evolution, 11: 257-277.

HUTCHEON J.M. & J.A.W. 2006: A moveable face: deconstructing the Microchiroptera and a new classification of extant bats, Acta Chiropterologica, 8 (1): 1-10.

IRWIN N. R., BAYERLOVÁ M., MISSA O. & MARTÍNKOVÁ N. 2012: Complex patterns of host switching in New World arenaviruses, Molecular Ecology, 21: 4137-4150.

JONES K. E., PURVIS A., MACLARNON A., BININDA-EMONDS O.R.P. & SIMMONS N.B. 2002: A phylogenetic supertree of the bats (Mammalia: Chiroptera), Biological Reviews, 77: 223-259.

KEARSE M., MOIR R., WILSON A., STONES-HAVAS S., CHEUNG M., STURROCK S., BUXTON S., COOPER A., MARKOWITZ S., DURAN CH., THIERER T., ASHTON B., MEINTJES P. & DRUMMOND A. 2012: Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data, Bioinformatics, 28 (12): 1647-1649.

KEANE T. M., CREEVEY C. R., PENTONY M. M., NAUGHTON T. J. & MCLNERLEY J. O. (2006): Assessment of methods for amino acid matrix

47 selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified. BMC Evolutionary Biology, 6:29.

LACK J. B., ROEHRS Z. P., STANLEY C. E., JR., RUEDI M. & VAN DEN BUSSCHE R. A. 2010: Molecular phylogenetics of Myotis indicate familial-level divergence for the genus Cistugo (Chiroptera), Journal of Mammalogy, 91(4): 976–992.

LANFEAR R., CALCOTT B., HO S. Y. W. & GUINDON S. 2012: PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses, Molecular Biology and Evolution, 29 (6): 1695–1701.

LANGWIG K. E., FRICK W. F., BRIED J. T., HICKS A. C., KUNZ T. H. & KILPATRICK A. M. 2012: Sociality, density-dependence and microclimates determine the persistence of populations suffering from a novel fungal disease, white-nose syndrome, Ecology Letters, 15: 1050–1057.

LIPSCOMB D. 1998: Basics of Cladistic Analysis, George Washington University, Washington D. C.

LIU K., LINDER C. R., WARNOW T. 2011: RAxML and FastTree: Comparing Two Methods for Large-Scale Maximum Likelihood Phylogeny Estimation, PLoS ONE, 6 (11): e27731.

LORCH J. M., METEYER C.U., BEHR M.J., BOYLES J.G., CRYAN P.M., HICKS A.C., BALLMANN A.E., COLEMAN J.T.H., REDELL D.N., REEDER D.M. & BLEHERT D.S. 2011: Experimental infection of bats with Geomyces destructans causes white-nose syndrome, Nature, 480: 376-U129.

MARTÍNKOVÁ N., BAČKOR P., BARTONIČKA T., BLAŽKOVÁ P., ČERVENÝ J., FALTEISEK L., GAISLER J., HANZAL V., HORÁČEK D., HUBÁLEK Z., JAHELKOVÁ H., KOLAŘÍK M., KORYTÁR L., KUBÁTOVÁ A., LEHOTSKÁ B., LEHOTSKÝ R., LUČAN R.K., MÁJEK O., MATĚJŮ J., ŘEHÁK Z., ŠAFÁŘ J., TÁJEK P., TKADLEC E., UHRIN M., WAGNER J., WEINFURTOVÁ D., ZIMA

J., ZUKAL J. & HORÁČEK I. 2010: Increasing incidence of Geomyces destructans fungus in bats from the Czech Republic and Slovakia, Public Library of Science ONE, 5(11): e13853.

MAYER F., DIETZ C. & KIEFER A. 2007: Molecular species identification boosts bat diversity, Frontiers in Zoology, 4(1): 239–255.

MILLER-BUTTERWORTH, C. M., MURPHY W. J., O'BRIEN S.J., JACOBS D.S, SPRINGER M.S. & TEELING E. C. 2007: A Family Matter: Conclusive Resolution of the Taxonomic Position of the Long-Fingered Bats, Miniopterus, Molecular Biology and Evolution, 24(7): 1553-1561.

MYERS P., SMITH J. D., LAMA H. & KOOPMAN K. F. 2000: A recent collection of bats from Nepal, with notes on Eptesicus dimissus, Zeitschrift für Säugetierkunde, 65: 149-156.

O’HARA R.J. 1998: Population thinking and tree thinking in systematics, Zoologica scripta, 26: 323-329.

PHILIPPE, H. & TELFORD M. J. 2006: Large-scale sequencing and the new animal

Phylogeny, Trends in Ecology and Evolution, 21:614–620.

PIKULA J., BANDOUCHOVÁ H., NOVOTNÝ L., METEYER C. U., ZUKAL J., IRWIN N. R., ZIMA J. & MARTÍNKOVÁ N. 2012: Histopathology Confirms White-Nose Syndrome in Bats in Europe, Journal of Wildlife Diseases, 48(1), 207–211.

PUECHMAILLE S. J., VERDEYROUX P., FULLER H., GOUILH M. A., BEKAERT M. & TEELING E. C. 2010: White-Nose Syndrome Fungus (Geomyces destructans) in Bat, France, Emerging Infectious Diseases, 16(2): 290-293.

PUECHMAILLE S. J., WIBBELT G., KORN V., FULLER H., FORGET F., MÜHLDORFER K., KURTH A., BOGDANOWICZ W., BOREL CH., BOSCH T.,

CHEREZY T., DREBET M., GӦRFӦl T., HAARSMA A., HERHAUS F., HALLART G., HAMMER M., JUNGMANN CH., LE BRIS Y., LUTSAR L., MASING M., MULKENS B., PASSIOR K., STARRACH M., WOJTASZEWSKI A., ZӦPHEL U. & TEELING E. C. 2011: Pan-European Distribution of White- Nose Syndrome Fungus (Geomyces destructans) Not Associated with Mass Mortality, Public Library of Science ONE, 6(4): e19167.

PUECHMAILLE S. J., WINIFRIED F. F, KUNZ T. H., RACEY P. A., VOIGT CH. C., WIBBELT G. & TEELING E. C. 2011: White-nose syndrome: is this emerging disease a threat to European bats?, Trends in Ecology and Evolution, 26(11): 570-576.

ROEHRS Z. P., LACK J.B. & VAN DEN BUSSCHE R. A. 2010: Tribal phylogenetic relationships within Vespertilioninae (Chiroptera: Vespertilionidae) based on mitochondrial and nuclear sequence data, Journal of Mammalogy, 91(5):1073–1092.

RONQUIST F., TESLENKO M., VAN DER MARK P., AYRES D. L., DARLING A., HÖHNA S., LARGET B., LIU L., SUCHARD M. A. & HUELSENBECK J. P. 2012: MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space, Systematic Biology, 61 (3): 539-542.

RYAN GREGORY T. 2008: Understanding Evolutionary Trees, Evolution: Education and Outreach, 1:121-137.

SIMMONS N. B. & GEISLER J.H.1998: Phylogenetic relationships of Icaronycteris, Archeonycteris, Hassianycteris and Palaeochiropteryx to extant bat lineages, with comments on the evolution of echolocation and foraging strategies in microchiroptera. Bulletin of the American Museum of Natural History, 235: 1-182.

SIMMONS N. B. 2005: Mammal Species of the World, a taxonomic and geographic reference, 3rd ed. WILSON D. E. & REEDER D. M., The Johns Hopkins University Press, Baltimore, Maryland.

SMITH A. T. & XIE Y. 2008: A Guide to the Mammals of China, Princeton University Press

SPITZENBERGER, F., STRELKOV, P. & HARING, E. (2003): Morphology and mitochondrial DNA sequences show that Plecotus alpinus Kiefer & Veith, 2002 and Plecotus microdontus Spitzenberger, 2002 are synonymus of Plecotus macrobullaris Kuzjakin, 1965, Natura Croatia, 12: 39–53.

STAMATAKIS A. 2006 : RAxML-VI-HPC: Maximum Likelihood-based Phylogenetic Analyses with Thousands of Taxa and Mixed Models, Bioinformatics, 22 (21): 2688–2690.

TAVARE S. 1986: Some Probabilistic and Statistical Problems in the Analysis of DNA Sequences, Lectures on Mathematics in the Life Sciences (American Mathematical Society), 17: 57–86.

TEELING E. C., MADSEN O., VAN DEN BUSSCHE R.A., DE JONG W.W., STANHOPE M.J. & SPRINGER M.S. 2002: Microbat paraphyly and the convergent evolution of a key innovation in Old World rhinolophoid microbats, Proceedings of the National Academy of Sciences of the United States of America, 99: 1431-1436.

TEELING E. C., SPRINGER M.S., MADSEN O., BATES P., O’BRIEN S.J. & MURPHY J.W. 2005: A Molecular Phylogeny for Bats Illuminates Biogeography and the Fossil Record, Science, 307:580-584.

UNITED STATES GEOLOGICAL SURVEY (USGS), National Wildlife Health Center 2013. < http://www.nwhc.usgs.gov/disease_information/white- nose_syndrome/index.jsp. > Accessed April 3, 2013.

U.S. FISH & WILDLIFE SERVICE, White-Nose Syndrome (A Coordinated Response to the Devastating Bat Disease). < http://whitenosesyndrome.org/about/bats-affected-wns.> Accessed February 26, 2013.

VAIDYA G., LOHMAN D. J., & MEIER R. 2011: SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information, Cladistics, 27: 171–180.

VAN DEN BUSSCHE R. A. & HOOFER S.R. 2004: Phylogenetic Relationships among Recent Chiropteran Families and the Importance of Choosing Appropriate Out-Group Taxa, Journal of Mammalogy, 85(2): 321-330.

VOLLETH M. & HELLER K. G. 1994: Phylogenetic relationships of vespertilionid genera (Mammalia: Chiroptera) as revealed by karyological analysis, Journal of Zoological Systematics and Evolutionary Research, 32: 11– 34.

WARNECKE L., TURNER J. M., BOLLINGER T. K., LORCH J.M., MISRA V., CRYAN P.M., WIBBELT G., BLEHERT D.S. & WILLIS C. K.R. 2012: Inoculation of bats with European Geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome, Proceedings of the National Academy of Sciences of the United States of America, 109(18): 6999-7003.

WEBB C. O., ACKERLY D. D. & KEMBEL S. W. (2008): Phylocom: software for the analysis of phylogenetic community structure and trait evolution, Bioinformatics, 24: 2098-2100.

WIBBELT G., KURTH A., HELLMANN D., WEISHAAR M., BARLOW A., VEITH M., PRÜGER J., GӦRFӦL T., GROSCHE L., BONTADINA F., ZӦPHEL U., SEIDL H-P., CRYAN P. M. & BLEHERT D. S. 2010: White-nose syndrome fungus (Geomyces destructans) in bats, Europe. Emerging Infectious Diseases, [serial on the Internet].

WILDER A.P., FRICK W.F., LANGWIG K.E. & KUNZ T. H. 2011: Risk factors associated with mortality from whitenose syndrome among hibernating bat colonies, Biology Letters, 7: 950–953.

WILLIS C.K.R.,1, MENZIES A. K, BOYLES J.G. & WOJCIECHOWSKI M. S. 2011: Evaporative Water Loss Is a Plausible Explanation for Mortality of Bats

52 from White-Nose Syndrome, Integrative and Comparative Biology, 51(3): 364– 373.