Discovery and Evolutionary Affinities of Five New Species of Amphibians from Bangladesh

Home , Batrachia, Blommersia, Dicroglossidae, Euphlyctis, Euphlyctis cyanophlyctis, Fejervarya

Discovery and evolutionary affinities of five new species of amphibians from Bangladesh

Mohammad Sajid Ali Howlader

Ecological Genetics Research Unit Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki Finland

Academic Dissertation To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences of the University of Helsinki in Latokartanonkaari 7 - B- rakennus, Luentosali 3 (A108) on 13th May 2016 at 12 noon.

Helsinki 2016

Supervised by: Prof. Juha Merilä Department of Biosciences University of Helsinki, Finland

Dr. Abhilash Nair Department of Biosciences University of Helsinki, Finland

Thesis advisory committee: Prof. Jyrki Muona Zoology Unit, Finnish Museum of Natural History University of Helsinki, Finland

Dr. Péter Poczai Botany Unit, Finnish Museum of Natural History University of Helsinki, Finland

Reviewed by: Dr. Risto Väinölä Zoology Unit, Finnish Museum of Natural History University of Helsinki, Finland

Dr. Ilari Eerikki Sääksjärvi Zoological Museum, Department of Biology University of Turku, Finland

Examined by: Associate Prof. Tommi Nyman Department of Biology University of Eastern Finland, Finland

Custos: Prof. Veijo Kaitala Department of Biosciences University of Helsinki, Finland

Author‘s address: Ecological Genetics Research Unit, Department of Biosciences, PO Box 65 (Biocenter 3, Viikinkaari 1), University of Helsinki, Finland E-mail: [email protected], [email protected]

Cover design by: Howlader, MSA ISBN 978-951-51-2105-9 (paperback) ISBN 978-951-51-2106-6 (PDF) Available at http://ethesis.helsinki.fi Unigrafia, Helsinki 2016.

Disclaimer: This publication (or thesis) is not available for purposes of zoological nomenclature in accordance with the International Code of Zoological Nomenclature (ICZN), article 8.3 (“If a work contains a statement to the effect that all or any of the names or nomenclatural acts in it are disclaimed for nomenclatural purposes, the disclaimed names or acts are not available. Such a work may be a published work….”).

Contents

ABSTRACT ...... 5 INTRODUCTION ...... 6 Conservation begins by species discovery ...... 6 Trends in species discovery: Amphibia ...... 7 Species and systematics under molecular evolutionary framework ...... 9 Historical perspective to amphibian systematics in South Asia ...... 10 Amphibian diversity and conservation challenges in Bangladesh ...... 11 Aims ...... 13 MATERIALS AND METHODS ...... 14 Taxa and specimens ...... 14 Morphological measurements ...... 15 Sequence analyses and phylogenetic methods...... 16 Divergence time estimation ...... 17 Bioacoustic analyses ...... 17 Ethics Statement ...... 17 RESULTS AND DISCUSSION ...... 18 Phylogenetic relationships of Fejervarya and Zakerana genera ...... 18 New Species ...... 20 Zakerana and Fejervarya ...... 20 Euphlyctis ...... 21 Microhyla ...... 22 Potential threats ...... 23 CONCLUSIONS AND FUTURE DIRECTIONS ...... 25 ACKNOWLEDGEMENTS ...... 26 REFERENCES ...... 27

The thesis is based on the following articles, which are referred to in the text by their Roman numerals:

I. Howlader MSA (2011) A new species of Fejervarya (Anura: Dicroglossidae) from Bangladesh. Zootaxa 2761: 41–50.

II. Howlader MSA, Nair A, Gopalan SV, Merilä J (2016) Molecular phylogeny of the genus Fejervarya (Anura: Dicroglossidae) with a description of new species from Dhaka. Submitted, manuscript.

III. Howlader MSA, Nair A, Merilä J (2016) A new species of frog (Anura: Dicroglossidae) discovered from the mega city of Dhaka. PLoS ONE 11: e0149597.

IV. Howlader MSA, Nair A, Gopalan SV, Merilä J (2015) A new species of Euphlyctis (Anura: Dicroglossidae) from Barisal, Bangladesh. PLoS ONE 10: e0116666.

V. Howlader MSA, Nair A, Gopalan SV, Merilä J (2015) A new species of Microhyla (Anura: Microhylidae) from Nilphamari, Bangladesh. PLoS ONE 10: e0119825.

Authors’ contributions I II III IV V

Conceived and designed the MSAH MSAH, JM, AN MSAH, JM, AN MSAH, JM, AN MSAH, JM, AN study

Performed the analyses MSAH MSAH, AN MSAH, AN MSAH, AN, JM MSAH, AN, JM

Contributed study material MSAH MSAH, SVG MSAH MSAH, SVG MSAH, SVG

Conduct the field work MSAH MSAH MSAH MSAH MSAH

Manuscript preparation MSAH MSAH, JM, AN MSAH, JM, AN MSAH, JM, AN MSAH, JM, AN,

Obtained permission for MSAH MSAH MSAH MSAH MSAH fieldwork and collection

Received research funding ------MSAH, JM MSAH, JM MSAH, JM MSAH, JM

MSAH: Mohammad Sajid Ali Howlader, AN: Abhilash Nair, SVG: Sujith V Gopalan, JM: Juha Merilä

ABSTRACT

Amphibians are the most threatened class of vertebrates. About 48% of the known amphibian species are threatened by extinction, and many species still remain undescribed, especially from tropical and sub-tropical countries such as Bangladesh. In contrast to India and Sri Lanka, amphibian diversity in Bangladesh is poorly known, and little effort has been put towards documenting the species diversity and resolving evolutionary affinities among amphibian taxa in this country. Hence, the actual diversity of amphibians in Bangladesh remains unknown. The aim of this dissertation work was to improve our knowledge of amphibian diversity in Bangladesh by identifying and describing new amphibian species and investigating their evolutionary relationships with closely related taxa.

I used morphological and molecular phylogenetic methods to identify and describe one new genus and five new species from different genera. In addition to using traditional morphological comparisons, I also utilized mitochondrial gene fragments to estimate phylogenetic affinities among the studied taxa, with Maximum-likelihood and Bayesian methods. The first two chapters of the thesis focus on the amphibian genera Fejervarya and Zakerana, the latter which was previously embedded within Fejervarya. These chapters also include descriptions of two new species – Fejervarya asmati sp. nov. (now Zakerana asmati), as well as Fejervarya burigangaensis sp. nov. 1, respectively. In the third chapter, a new species (Zakerana dhaka sp. nov.) is described from the urban core of Dhaka, the capital of Bangladesh and one of the most densely populated mega cities in the world. In the fourth chapter, I describe Euphlyctis kalasgramensis sp. nov., which was earlier recognized as E. cyanophlyctis, and show that it is genetically highly divergent from the E. cyanophlyctis described from southern India. In the last and fifth chapter, I describe Microhyla nilphamariensis sp. nov. as a new species. It is a member of a highly genetically heterogeneous group of frogs that have been recognized as M. ornata for the past 173 years. In general, the results of the studies included in this dissertation advance our understanding of amphibian diversity in Bangladesh and adjacent regions, and show that discovery and description of new amphibian species from this region is still fairly easy. Consequently, it seems likely that more thorough sampling and further investigations in this region can uncover additional new amphibian species to science. Such studies, together with the discoveries described in this thesis, should also provide useful information for understanding and conserving the amphibian biodiversity in this poorly studied region.

1 Provided scientific name is not available for purposes of zoological nomenclature in accordance with the International Code of Zoological Nomenclature (ICZN), article 8.3. Chapter (II), containing this scientific name is in the publication process.

Summary

including mankind. Given these facts, it is INTRODUCTION not surprising that amphibians have experienced higher extinction rates and more dramatic population size declines Many studies suggest that the “sixth than most other vertebrate groups mass extinction” is under way (Pereira et (Stuart et al. 2004; Hoffmann et al. 2010). al. 2010; Barnosky et al. 2011; Alroy There are a number of suggestions as to 2015; McCallum 2015). With estimated how amphibian species could be saved extinction rates between 0.01 and 1% from the predicted mass extinction, such per decade, 500 to 50000 species are as the 11 priority action plans identified predicted to face extinction per decade by IUCN (e.g. identifying priority under assumption that there are 5 conservation sites, securing existing million species on Earth (Costello et al. habitats, captive breeding, detection and 2013). Extinction rates of vertebrates control of infectious disease etc.; Gascon are likely to be comparable to the mass et al. 2007). Research on systematics and extinctions that have taken place in the taxonomy are also listed among the IUCN geological past (McCallum 2015). The priorities (Gascon et al. 2007). However, threat of extinction is particularly acute some of these conservation actions are in the case of amphibians (Wake and difficult to implement effectively, Vredenburg 2008; Hoffmann et al. 2010). especially in areas where the true Already, about 48% of the known species diversity is still unknown. amphibian species are under threat of Obviously, systematic work and species extinction, which is a much higher figure discovery are important steps toward than in other vertebrate classes (Stuart effective conservation of species (Köhler et al. 2004). The main drivers of these et al. 2005). Only after a species has been extinctions are habitat destruction, described, studies focused on agrochemicals and chemical pollution, distribution, abundance as well as on several emerging infectious diseases, genetic diversity can be initiated introduced species, exploitation, and (Costello et al. 2013). climate change (Beebee and Griffiths

2005). Hence, many of these reasons can be directly or indirectly attributed to human activities (Pounds 2001; Conservation begins by species Davidson et al. 2002; Riley et al. 2005). discovery Amphibians have permeable, moist, and thin skins (Elkan and Cooper 1980), Global attention has been focused on making them particularly vulnerable for conserving rare and threatened desiccation (Rohr and Madison 2003) amphibian species and their habitats and environmental pollutants (Blaustein (Wake and Vredenburg 2008; Barnosky et al. 2003). Moreover, their bi-phasic life et al. 2011). The ongoing conservation history which involves life stages that efforts to reduce biodiversity loss have are first aquatic and later terrestrial led to prioritizing biodiversity hotspots exposes them to different challenges (e.g. Madagascar, Sri Lanka, Western faced in each habitat (Becker et al. 2010; Ghats etc.), where a large number of Amburgey et al. 2012; Fonseca et al. endemic amphibian species are faced 2013). This sensitivity of amphibians to with habitat loss (Meegaskumbura et al. environmental perturbations makes 2002; Gunawardene et al. 2007; Vieites them an early-warning system for et al. 2009). Obviously, the task of predicting changes that may eventually conservation cannot be implemented influence more resistant species, unless the species diversity is well

documented. How should conservation Trends in species discovery: deal with those species which are still unknown? IUCN (2016) recently Amphibia assessed the threat status of around The global species diversity remains 6408 amphibian species (from ~7400 poorly described; it has been estimated described species; AmphibiaWeb 2015a) that about 86% of existing species on of the world. However, there are still earth still await to be described (Mora et more species to be described (e.g. al. 2011). However, during the last 30 Meegaskumbura et al. 2002; Bossuyt et years, the total number of known al. 2004), many of them being cryptic amphibian species has increased by over species recognized currently under the 60% (AmphibiaWeb 2015a), and a wrong species name (e.g. Nair et al. number of new species have been 2012). For example, at least 14 new frog described even from places where their species were described from only two discovery was unexpected. For example, nominal species (Odorrana livida and Rana kauffeldi was described from the Rana chalconota) in Southeast Asia New York metropolitan area in 2014 (Stuart et al. 2006). Such cryptic species (Feinberg et al. 2014). Like this may lead to an underestimation of the discovery, a number of species are also current threat status of species in the being regularly described from other IUCN Red list: when a formerly well-studied areas. Ironically, the recognized species with a wide number of known amphibian species has geographic distribution is split into been increasing despite the fact that several distinct species, each of the new amphibians exhibit the highest species are likely to have a smaller extinction rate among all vertebrates population size than previously (Stuart et al. 2004). This may mean that anticipated, and also, face different many amphibian species are possibly threats than the formerly recognized, extinct before they are even described more widespread taxon. Therefore, (Costello et al. 2013). In this view, basic separate conservation measures may be systematic and taxonomic work is required for each of the new species, important for both conservation and which may also differ in their ecologies. management (Köhler et al. 2005), not Furthermore, incomplete information only of rare and threatened amphibian about species diversity may lead to species, but also of common species. The oversights regarding biodiversity current number of known amphibian hotspots (Köhler et al. 2005), as well as species exceeds 7400, and only within mislead identification of priority areas the last eleven years (2004 - 2014), 1769 for conservation. In general, species new amphibian species have been discovery inspires the identification of described (AmphibiaWeb 2015b). This areas with the highest concentrations of high rate of increase in vertebrate species and their conservation (Bickford species number reflects the keen et al. 2007). Hence, discovery of new scientific interest towards species species should be of high priority for discovery (Hanken 1999). This recent global conservation policy (Köhler et al. proliferation of newly described 2005; Bickford et al. 2007; Costello et al. amphibian species also likely reflects the 2013). recent incorporation of molecular methods to traditional phenotypic methods in systematics and alpha taxonomy. Molecular phylogenetics has

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Summary

Box 1. What is a species?

There are many definitions for “species”, but there is no universal species concept that is applicable to all organisms. Each of the different species concepts emphasizes different criteria and differ from each other in biological reasoning. For example, the biological species concept places attention on the property of reproductive isolation (Mayr 1942, 1970; Dobzhansky 1970), whereas the ecological species concept highlights the importance of the occupation of a distinct niche or adaptive zone (Van Valen 1976; Andersson 1990). One version of the phylogenetic species concept advocates diagnosability of the smallest cluster of individuals within which there is a parental pattern of ancestry and descent (Cracraft 1983; Nixon and Wheeler 1990). Another version of the phylogenetic species concept advocates monophyly (Rosen 1979; Donoghue 1985). According to the genotypic cluster species concept, a species is considered distinct if the samples form a single cluster in the frequency distribution of multilocus phenotypes and genotypes (see in: Mallet 1995). The morphological species concept is a classical concept that has been used since Aristotle and Linnaeus: this concept emphasizes species as a groups of organisms with consistently and persistently distinct trait(s) (Cronquist 1978). The diversity of species concepts is not very surprising since biologists have diversified interests; systematists tend to emphasize diagnosability and monophyly of species, ecologists favour niche differences, paleontologists and museum taxonomists tend to highlight morphological differences, and population geneticists and molecular systematists tend to prioritize genetic criteria, and biologists who study hybrid zones give importance to reproductive barriers (De Queiroz 2005). The figure above illustrates some of the criteria employed by different species concepts. In this figure, all of these species concepts are different from each other, but they all serve the single objective to define a basic unit of biological classification: the species. In this thesis, I have combined morphological and phylogenetic concepts to define five new species of amphibians (see text). allowed identification of sequence data, which comes mostly from morphologically “cryptic” new mitochondrial protein-coding or amphibian species (e.g. Meegaskumbura ribosomal genes, for constructing and Manamendra-Arachchi 2005; Stuart molecular phylogenies to position taxa et al. 2006). Current molecular (Pyron 2015). Specifically, molecular systematics of amphibians relies on DNA identification of evolutionarily

independent lineages of taxa has allowed universal yardstick to define species a large number of morphologically (Wiley 1978; Mishler and Donoghue cryptic species having overlapping 1982). For example, the biological (sympatric) or contiguous (parapatric) concept is simply unsuitable for asexual geographic ranges to be identified (e.g. organisms (Mayr 2000). The biological Chapters IV and V). species concept also meets serious difficulties in situations where However, major advances in hybridization occurs, as is the case for amphibian systematics have also been many plants (Donoghue 1985). On the accomplished by phenotypic approaches other hand, with the recently increased based on the nomenclatural and application of molecular tools to literature-based sorting that dominated systematic problems, the biological the last century (Dubois 1980a; 1981; species concept has been challenged by 1984b; 1986). This work has helped alternative species definitions (Bickford establish a solid foundation for modern et al. 2007), such as phylogenetic and molecular based systematics. On the evolutionary species concepts (e.g. other hand, bioacoustics analysis (e.g. Simpson 1961; Cracraft 1983; Nixon and Andreone et al. 2010; Qin et al. 2015) and Wheeler 1990). These alternative ecology (e.g. Andreone et al. 2010) are species concepts put less emphasis on also integrated with modern amphibian reproductive isolation as the theoretical species discovery, which, together with and practical standard to define species morphological and molecular methods, (Meier 2000). Instead, they emphasize have allowed further discovery of new phylogenetic history and distinct species. In particular, distinctive male evolutionary lineages or units as distinct calls can be especially indicative of pre- species (Box 1). mating reproductive isolation (Kelley 2004), and therefore serve as an efficient Recently, the phylogenetic tool for new species identification. framework has become adopted for classification of many organisms at higher levels of taxonomic hierarchy (e.g. Bininda-Emonds et al. 2007; Pyron and Species and systematics under Wiens 2011; Betancur-R et al. 2013; molecular evolutionary Pyron et al. 2013; Burleigh et al. 2015). framework The general understanding of amphibian phylogeny advanced dramatically from Species is the basic unit of organismal the late 1960s to the early 1980s (Frost classification, but its definition remains et al. 2006), and much credit for this goes the source of continued controversy to earlier work that laid important (Meier 2000; Box. 1). Before the end of foundations (e.g. Inger 1967; Kluge and the last century, the biological species Farris 1969; Lynch 1971; Lynch 1973). concept advocated by Mayr (1942, 1970) However, it is only during the last decade was popular. According to this definition, that large-scale molecular phylogenies of species are groups of interbreeding, or amphibians have become available potentially interbreeding natural (Frost et al. 2006; Pyron and Wiens populations that are reproductively 2011). Some researchers advocate a isolated from other such groups (Mayr threshold percentage of genetic 1970). However, the mechanisms of divergence to designate distinct reproductive isolation vary among taxa, amphibian species (Vences et al. 2005; and the biological concept bids no Fouquet et al. 2007; Xia et al. 2012). For

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Summary instance, populations that show a high first binomial scientific name “Caecilia degree of genetic divergence on mtDNA glutinosa” (now Ichthyophis glutinosus) sequences exceeding a threshold level for a South Asian amphibian (Linnaeus (commonly ca. 3%; Fouquet et al. 2007; 1758). The systematic work on South Vieites et al. 2009) are treated as Asian amphibians by Schneider was putative species or "candidate" species followed by other European workers (e.g. Fouquet et al. 2007; Vieites et al. (e.g. Jerdon 1853; Günther 1860, 1876; 2009; Xia et al. 2012). Using Boulenger 1882, 1888, 1904, 1906, phylogenetic methods, species are 1919; Annandale 1909, 1912, 1913; defined based on monophyly, as it is Parker 1934), as well as by few native difficult to diagnose if these Indian (e.g. Rao 1922, 1937; Seshachar monophyletic groups are reproductively 1939) naturalists until India gained its isolated (Bickford et al. 2007). Many independence in 1947. After this, authors are also using morphological systematics and species discovery of and ecological data to support genetic amphibians in South Asia was advanced inferences in describing new species (e.g. by a series of successive contributions Andreone et al. 2010; Qin et al. 2015). from India (e.g. Pillai 1977, 1979, 1986; The research presented in this thesis Chanda 1990; Dubois 1975b, 1980b, generally applies phylogenetic and 1983), Pakistan (e.g. Dubois and Khan morphological species concepts to 1979; Khan and Tasnim 1989), Sri Lanka identify morphologically distinct new (e.g. Manamendra-Arachchi and species (Chapters I, II, III, IV and V). Pethiyagoda 2005), Nepal (e.g. Dubois New species are also distinguished from 1974, 1975a, 1984a) and Bhutan (e.g. congeners using comparison of genetic Delorme and Dubois 2001). The divergences (Chapters II, III, IV and V) remarkable discovery of 27 new species and bioacoustic differences (Chapters I from Sri Lanka by Manamendra- and II). In fact, both genetic divergence Arachchi and Pethiyagoda (2005) was and bioacoustic distinctiveness are mostly based on phenotypic correlated with the degree of post- comparisons. In contrast to these mating and pre-mating reproductive developments, the contributions to isolation, respectively (e.g. Kelley 2004; amphibian diversity from other South Malone and Fontenot 2008), which is Asian areas are modest. For instance, central to the biological species concept. only a single species [Bufo melanostictus (=Duttaphrynus melanostictus)] has been described from the Maldives on the basis Historical perspective to of historical collections done in 1901 (Glaw and Rosado 2006). In Bangladesh, amphibian systematics in South Hylorana tytleri is the single valid species Asia described from the city of Dhaka [="Dacca", Theobald (1868)] before The foundation for systematic work on 2011 (see below). amphibians in South Asia was laid by German naturalist Johann Gottlob Though traditional phenotypic Schneider in 1799 (Schneider 1799), methods have dominated species who described many amphibian species discovery and systematics in South Asia (e.g. Duttaphrynus melanostictus, until the 21st century, they have been Euphlyctis cyanophlyctis, Sphaerotheca deemed insufficient in recognizing the breviceps, Uperodon systoma). Before actual species diversity in this region, this, Carl Linnaeus had designated the where a large number of cryptic species

go unidentified (Biju et al. 2014a). In 2003, the Western Ghats of India was put into the lime-light by the discovery of a new frog family (Nasikabatrachidae) having an ancient biogeographical link with Africa, as revealed by molecular phylogenetic methods (Biju and Bossuyt 2003). After 2003, the application of molecular phylogenetic methods have quickly doubled the overall species number in South Asia, especially in India (e.g. Biju and Bossuyt 2009; Kamei et al. 2009; Bocxlaer et al. 2011; Zachariah et al. 2011; Kamei et al. 2012; Biju et al. 2014a, 2014b) and Sri Lanka (Meegaskumbura and Manamendra- Arachchi 2005; Meegaskumbura et al. 2009). Most recently, mitochondrial DNA-based phylogeny has revealed 14 new species in the genus Micrixalus, which were earlier lumped together in ~7 previously known species (Biju et al. 2014a). However, species discovery with molecular methods in Pakistan, Nepal, Bhutan and Bangladesh has not progressed in parallel to that in India and Sri Lanka.

Amphibian diversity and conservation challenges in Bangladesh

Bangladesh is one of the most densely populated countries in the world (World Bank 2015), but little is known about amphibian diversity in this country. In fact, herpetology in Bangladesh has been neglected for the past century (Molur 2008; Fig. 1). Before modern times, only a single species of amphibian has been Fig. 1. Timeline depicting history of amphibian diversity described from Bangladesh (formerly: in Bangladesh. Red line shows the total number of East Bengal, and East Pakistan) in 1868 species recognized by year, whereas the blue line depicts by naturalist William Theobald (1868). the number of new species recognized in a given year. After a long period of political instability following British ruling and separation from Pakistan (in 1971), the study of was resumed by Husain and Rahman amphibian systematics in Bangladesh (1978). The number of valid amphibian

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Summary

Fig. 2. Schematic illustration of habitat types in Bangladesh from where the new species described in this thesis were discovered. The upper left corner contains a graph of the total number of species, average rainfall and temperature for each month. species (not misidentified or not known and unknown amphibian species. synonymized under another species) in About eight amphibian species of Bangladesh has ranged from 9 to 13 until Bangladesh were considered to be 2003 (Fig. 1) when Asmat et al. (2003) threatened by IUCN in the year 2001 made locality records of a few known (Islam et al. 2000), but the current state Asian species from Chittagong hill tracts of affairs might be even worse. While region. Since then, the number of local legal acts (e.g. former “Bangladesh amphibian species encountered from Wildlife Act 1974”, and newly reformed Bangladesh has increased (Fig. 1), with “Wildlife Preservation & Security Acts, 31 species currently listed (e.g. Khan 2012”) have significant roles in 1997a; Asmat et al. 2003; Rasel et al. protecting large mammals found in 2007; Reza and Mahony 2007). Before protected forests (Khan 2004), there are Chapter I was published, all studies no legal acts directed towards reporting new amphibian species from amphibians, although they are widely Bangladesh were based on recording consumed (e.g. Niekisch 1986) and used species that were already discovered as live bait for fishing (Fig. 2). earlier from neighboring countries (e.g. Furthermore, people living close to the India, Nepal): the species described in protected areas often disobey the the Chapter I is the first discovery of a conservation measures taken by local new species from Bangladesh (see in: government (Sarker and Røskaft 2011). Fisher 2011) since 1868. While the Therefore, amphibians of Bangladesh are number of amphibian species in in need of monitoring and conservation Bangladesh has increased very slowly planning, but this is very challenging as (Fig. 1), the rate of habitat loss and the actual species statuses and diversity destruction in Bangladesh has been very are still unknown (see above, Fig. 1). To rapid; about 2600 ha of forest is this end, molecular tools may provide disappearing every year (Nandy et al. the most efficient and effective way to 2013), possibly threatening many document the amphibian species

Fig. 3. A flowchart summarizing the approaches used to investigate the systematic relationships among the new amphibian species discovered in this thesis. Roman numerals (I –V) refer to the five chapters of this thesis.

diversity in Bangladesh (e.g. Chapters II, species are already formally published as III, IV and V; Hasan et al. 2012b, 2014b). per ICZN rules. All of these species were, and hence, not recognized as distinct Aims species for regional conservation efforts. Furthermore, I investigated evolutionary The aim of this thesis was to fulfill some affinities of two genera (Fejervarya of the knowledge gaps in our sensu stricto and Zakerana) which have understanding of amphibian species been subject to much scientific debate diversity in Bangladesh, as well as to and controversy recently. shed light on evolutionary affinities among amphibian taxa. To this end, specimens of five unidentified species were collected from unprotected sites in Bangladesh, from which mtDNA was sequenced and used in phylogenetic analyses (Fig. 3). Five cryptic lineages from four South Asian genera were discovered and formally described as new species to science using both molecular and traditional taxonomic approaches (Fig. 3). Four of the new

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Summary

MATERIALS AND METHODS and Euphlyctis). The specimens were collected from Chittagong University campus (Chapter I), Shere-Bangla Nagar Taxa and specimens (Chapters II and III), Kalasgram The specimens used in my studies (Chapter IV), and Saidpur (Chapter V) included individuals of four different in Bangladesh (Fig. 4). All used genera (Fejervarya, Zakerana, Microhyla,

Fig. 4. A map showing the geographic locations of the sites from where the type specimens for the new species described in this thesis were collected. Roman numerals refer to chapters of this thesis.

specimens, including the type material, were deposited at the Finnish Museum of Natural History, Helsinki, Finland (MZH) and Zoology Department, University of Chittagong, Bangladesh (MZD). Additional specimens used for morphological comparisons were examined from various museums, including the Finnish Museum of Natural History, Finland (MZH), Rajiv Gandhi Centre for Biotechnology, Kerala, India (RGCB), Zoology Department, University of Chittagong, Bangladesh (MZD), Museum of Herpetology Fig. 5. Schematic illustration of the morphological measures Laboratory Bangladesh, Ichamoti definitions used in the chapters of this thesis. Trait abbreviations are explained in Materials and Methods. college, Dinajpur, Bangladesh (MHLB), and Zoological Survey of India (ZSI). Accession numbers of all used specimens webbing formula followed that of Glaw are provided in individual chapters. and Vences (2007).

Morphological comparisons were Morphological measurements done using both ratios and actual measurements. Ratios were used since

morphological differences within some Both qualitative (e.g. coloration and of the studied genera mainly involve tubercle arrangements) and quantitative differences in body proportions (Veith et characters were used throughout this al. 2001; Kuramoto et al. 2007). Ratios thesis. Quantitative measures were also allowed me to make direct taken with digital calipers, with accuracy comparisons to published data of to the nearest 0.02 mm (Chapters I, II, congeneric species for which the actual III, IV and V). Quantitative characters morphological data was missing (e.g. measured included (Fig. 5): SVL (snout- Parker 1934; Joshy et al. 2009; Hasan et vent length), HL (head length), HW (head al. 2012b, 2014b). However, I note that width), MN (distance from back of body proportions can be highly variable, mandible to nostril), SL (snout length), even among different populations of the MFE (distance from back of mandible to same species (e.g. Alho et al. 2011). front of eye), MBE (distance from back of Therefore, whenever sufficient data was mandible to back of the eye), IN available, multivariate statistical (internarial distance), IOD (interorbital analyses utilizing linear measurements distance), EN (distance from front of were used to compare the newly eyes to nostril), NS (nostril–snout described species and their length), EL (eye length), UEW (maximum phylogenetically and morphologically width of upper eyelid), TD (tympanum closely related congeners. For these diameter), TEL (tympanum–eye length), analyses, I obtained morphological HAL (hand length), FAL (forearm measurements from museum specimens length), THIGHL (thigh length), TL (tibia (Chapters II, III, IV and V). These length), TFOL (length of tarsus and foot), multivariate statistical analyses included FOL (foot length). Descriptions of principal component analyses (PCA) and

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Summary discriminant function analyses (DFA). Gene sequences of 16S rRNA Simple bivariate scatterplots were also (Chapters II, III, IV and V), 12S rRNA used to further explore and exemplify (Chapters II, III and IV), Tyr (Chapter the morphometric differences II), RAG1 (Chapter II), RAG2 (Chapter (Chapters II and V) among the species. II), NCX1 (Chapter II), CXCR4 (Chapter One-way ANOVAs followed by Tukey’s II), and BDNF (Chapter II) for all the HSD tests were used to test if the PC- known species in the genera Fejervarya, scores differed significantly among Zakerana, Euphlyctis, Microhyla and species (Chapters II and V). All other related species were obtained statistical analyses were performed from the GenBank repository. Sequences using JMP Pro 10.0.2 software (SAS were aligned using ClustalW as Institute Inc., Gary, USA). implemented in BIOEDIT (Thompson et al. 1994; Hall 1999). Pairwise genetic distances between the Euphlyctis species Sequence analyses and were calculated using Mega v 5.5.6 (Tamura et al. 2011) excluding the sites phylogenetic methods with indels. The phylogenetic analyses were performed using Maximum For the genetic analyses, genomic DNA Likelihood (ML) and Bayesian inference was extracted from muscle tissues using methods. The GTR + I + G substitution a silica-based method (Ivanova et al. model was the most fitting nucleotide 2006) and stored at -20°C. PCR substitution model for the combined amplification and sequencing of two dataset and was used for ML analyses. mitochondrial DNA fragments (12S For the ML analysis, branch support was rRNA and 16S rRNA genes) was done evaluated by using 1000 bootstrap using three pairs of primers listed in replicates (Felsenstein 1985) as Chapters II, III, IV and V. PCR reaction implemented in Mega v 5.5.6 (Tamura et mix for both genes consisted of 5.72 μl of al. 2011). The Bayesian analyses were dH2O, 2 μl of 5x buffer, 0.08 μl of dNTP, conducted using MrBayes 3.1.2 0.2 μl of Phire enzyme (Thermo Fisher) (Ronquist and Huelsenbeck 2003). The and 0.5 μl of each primer, in a total analyses were performed as a reaction volume of 10 μl. The PCR partitioned dataset with each gene program started with a preliminary fragment having its own nucleotide denaturation step at 98°C for 30s, substitution model (see in: Chapters I, followed by 34 cycles of 98°C for 10s, II, III, IV and V). The Markov chain Monte 55°C for 10s, 72°C for 30s and final Carlo runs were done for partitioned extension at 72°C for 1 min. PCR dataset for 1 million generations with products were purified by using ExoSap sampling frequency of 100 and with each IT (USB Corporation, Cleveland, OH, partition unlinked for the substitution USA) and sequenced at the Institute for parameters. Convergence of the runs Molecular Medicine Finland (FIMM). was assessed by the average split Sequence ambiguities were edited frequency of standard deviations (<0.01) manually by aligning forward and and by checking the potential scale reverse reads using the Geneious 5.6.5 reduction factors (~ 1.0) for all model program (Drummond et al. 2013). parameters. The first 25% of trees was Obtained sequences were deposited in discarded as burn-in, and the remaining GenBank and accession numbers are trees were used to generate the 50% provided in individual chapters (II, III, majority rule consensus tree and to IV and V).

estimate the Bayesian posterior (Chapter I) were recorded using a probabilities. Canon Digital camera (model: IXY DIGITAL 10) in video mode, whereas a Sony Cyber-shot camera (model: DSC- Divergence time estimation W530) was used for Zakerana dhaka sp. nov. (Chapter III). Air temperature The divergence time estimation between during the recordings was between 23°C the Microhyla species (Chapter V) was and 24°C, when the calls were recorded done by generating a time tree as in the type localities of both species (Fig. implemented in the program BEAST 3). 1.8.1 [http://beast.bio.ed.ac.uk/]. The time tree was calibrated by introducing Calls of adult males (holotypes for two nodal constraints that correspond both species) were analyzed with the to: (a) M. mymensinghensis separation acoustic software Adobe Audition 3.0 from M. fissipes before 10.53 (5.48– [following (Rosa et al. 2010; Rosa and 16.95) mya (Hasan et al. 2014b) and (b) Andreone 2010)] and compared to the 1.7 million year old fossil series from the described bioacoustic data of Zakerana genus Gastrophryne (Family: species available in literature (Dubois Microhylidae; Sanchiz 1998; Holman 1975a; Dubois 1984a; Grosjean 2011). 2003). In the latter case, a normal Recordings were re-sampled at 44.1 Hz distribution with standard deviation of and 16 bit resolution in the mono pattern 0.5 was used to constrain the node and in “Waveform” extension. Frequency leading to G. olivacea and G. information was obtained through Fast mazatlanensis as having occurred Fourier Transformation (FFT, width between 0.72 and 2.68 mya. This 1024 points); the audio spectrogram was calibration point was used because many obtained at Hamming window function fossils of G. olivacea and G. mazatlanensis with 256 bands resolution. Call have been reported from Pleistocene properties were measured as defined by deposits ranging from 0.24 to 1.8 mya Cocroft and Ryan (1995), Köhler (2000) (Holman 2003). The divergence time and and Martins and Jim (2003). Mean, node ages were estimated using a standard deviation and range (as well as lognormal relaxed molecular clock in a number of analyzed units, n), of call Bayesian framework. Markov chain parameters with temporal Monte Carlo analyses were run for ten measurements in seconds (s) or million generations, sampled every 1000 milliseconds (ms) are provided. Mann generations. We used Tracer 1.5 Whitney U-tests were used to compare [http://beast.bio.ed.ac.uk/Tracer] to Zakerana dhaka sp. nov. with view the BEAST 1.8.1 output and to phylogenetically closely related Z. asmati verify that all parameters were (Chapter III). adequately sampled (effective sample sizes > 200). A burn-in of 1000 was selected before summarizing the time Ethics Statement trees. All the research in this thesis was conducted with the appropriate Bioacoustic analyses permissions (CCF letter no. 22.01.0000.101.23.2012.681 for Bioacoustic analyses were used in collecting specimens, CF memo no. Chapters I and II. Z. asmati calls 22.01.0000.101.23.2012 for transport)

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Summary and following guiding principles from Fejervarya sensu lato are paraphyletic the Forest Department, Ministry of with respect to another South Asian Forest and Environment, People’s genus, Sphaerotheca (Kotaki et al. 2008; Republic of Bangladesh, responsible Kotaki et al. 2010). I characterized and authority for wildlife research in the renamed a South Asian species group study areas. The collection and research (Howlader 2011) formerly included into protocols were approved by the Fejervarya sensu stricto as Zakerana, on committee of the Wildlife Section of the the basis of paraphyly of Fejervarya Forest Department, Bangladesh, and reported in other studies (e.g. Frost et al. fulfilled by all ethical conditions as 2006; Kotaki et al. 2008; Kotaki et al. dictated by the authority, and the law of 2010). Wildlife Preservation & Security Acts, 2012 (Chapter 10, section 48). Collected Recently, Dinesh et al. (2015) re- specimens are not threatened species analyzed molecular genetic information and they are not listed in IUCN Redlist or from Zakerana and Fejervarya sensu by CITES. None of the samples used in stricto, and after arriving at different this thesis were collected from protected conclusions than others (Kotaki et al. areas. 2008; Kotaki et al. 2010; Hasan et al. 2014a) proposed to synonymize Zakerana under Fejervarya sensu lato. In Chapter II, I re-analyzed data from RESULTS AND DISCUSSION Dinesh et al. (2015) by correcting errors in their use of the primary data (Kotaki Phylogenetic relationships of et al. 2008; Kotaki et al. 2010; Hasan et Fejervarya and Zakerana al. 2014a). My analyses (Chapter II), which included a total 5462 bp of genera sequences from eight genes (16S rRNA,

Bolkay (1915) described Fejervarya as a 12S rRNA, Tyr, RAG1, RAG2, NCX1, subgenus of Rana that is characterized CXCR4, and BDNF), indicated possible by a lack of lateral skin folds, and by paraphyly of Fejervarya sensu lato with skeletal features such as “omosternum Sphaerotheca being embedded in split forked”. As such, three species between Fejervarya sensu stricto and designated as Rana (“R. tigrina Daud., R. Zakerana; this was also found by Kotaki limnocharis Wiegm. und R. hexadactyla et al. (2008, 2010) and Hasan et al. Less.”; Bolkay 1915) actually bear (2014a). However, although the branch characteristics of Fejervarya. These containing Sphaerotheca, Fejervarya species are now placed in three different sensu stricto and Zakerana is well genera, as Hoplobatrachus tigerinus, supported (97% bootstrap and 1 Fejervarya limnocharis and Euphlyctis posterior probability support), the hexadactylus (Frost 2016). Fejervarya position of Sphaerotheca as a sister clade limnocharis was subsequently of Fejervarya sensu stricto is still poorly designated as the single type species of supported (50% bootstrap and 0.54 this genus (Dubois 1981). Since Bolkay posterior probability support; Chapter (1915), Fejervarya are thought to be II). In this respect, my results are similar distributed throughout Asia. Howlader to those of Kotaki et al. (2010) who found (2011) designated a new genus the highest support for position of (Zakerana) on the basis of phylogenetic Sphaerotheca as sister to Fejervarya studies which suggested that some South sensu lato. Interestingly, Hasan et al. Asian and Southeast Asian species of (2014a) did not find any statistical

Fig. 6. Photographs of new species that have already been published and included into this thesis. (A) Zakerana asmati, (B) Zakerana dhaka, (C) Euphlyctis kalasgramensis, and (D) Microhyla nilphamariensis. Photograph of another new species available in Chapter II, which is not yet published.

support for this, albeit the support value synonymizing Zakerana and Fejervarya was not provided. Likewise, Pyron and sensu lato, along with the fact that they Wiens (2011) found support for the cannot be morphologically diagnosed monophyletic grouping of the Fejervarya (Dinesh et al. 2015). sensu stricto and Zakerana clade, with Sphaerotheca as sister group. This is Recently, Ohler et al. (2014) particularly noteworthy in light of the presented results of principal fact that the data underlying their component analyses of morphological analysis came mostly from the same traits among Fejervarya sensu stricto, source (e.g. Kotaki et al. 2008; Kotaki et Minervarya, Sphaerotheca and Zakerana. al. 2010) as used in my analysis. My These analyses did not reveal any results did not recover the strong significant differences between monophyly of Zakerana and Fejervarya Fejervarya sensu stricto and Zakerana, sensu stricto as shown by Dinesh et al. whereas Minervarya and Sphaerotheca (2015) even though I was using the same differed significantly from each other underlying data. The monophyly of and also from Fejervarya sensu stricto Zakerana and Fejervarya sensu stricto and Zakerana. Based on these findings, was provided as the reason for Ohler et al. (2014) suggested that

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Zakerana be treated as a subgenus of snout length 0.67% of internarial Fejervarya sensu lato. However, the distance; MBE 18% of HL (Chapter I). I analyses of Ohler et al. (2014) did not also found that Z. asmati has an include all known species of Fejervarya advertisement call distinct from its sensu stricto (one out of 16) and geographical congeners. In Chapter III, I Zakerana (6 out 20), and used very few sequenced 16S and 12S rRNA genes of Z. specimens per species. asmati, which revealed that the species has the closest relationship with Taken together, both the Zakerana dhaka sp. nov. molecular genetic and phenotypic evidence in respect to the existence of distinct Fejervarya sensu stricto and My phylogenic analyses revealed Zakerana genera is conflicting, and that samples collected from Dhaka hence, the issue remains unresolved. The formed a distinct lineage within the phylogenetic relationship among Zakerana clade. This new lineage Fejervarya sensu stricto, Sphaerotheca clustered with Z. asmati with 99% and Zakerana can be only resolved with bootstrap support and posterior more extensive sequencing and probability of one (Chapter III). Both Z. morphological analyses including asmati and Zakerana dhaka sp. nov. additional taxa and specimens. formed a well-supported (97% bootstrap and 1 posterior probability support) sister group to Z. granosa and Z. New Species pierrei from Nepal and Bangladesh, respectively. Genetic divergence Zakerana and Fejervarya between Zakerana dhaka sp. nov. and other Zakerana species was very high (5 Zakerana is a highly diverse genus – 20.1% for 12S rRNA, and from 3.1 – occurring all over South Asia, and is 17.3 % for 16S rRNA). Bioacoustically, Z. composed of 20 recognized species asmati and Zakerana dhaka sp. nov. (Howlader 2011; IUCN 2016). In differed significantly from each other by Bangladesh, four species of Zakerana duration of inter-note intervals (W = (formerly Fejervarya sensu lato) have 550; p=0.001; Z. asmati, x = 56.25 ± been reported before 2011 (Rasel et al. 12.63, n=4; Zakerana dhaka sp. nov., x = 2007). In Chapter I, Z. asmati 32.4 ± 6.7, n =140), dominant frequency (“Fejervarya asmati”) was described (W = 458; p < 0.05; Z. asmati: 4100 – from the Chittagong (Bangladesh) by 5100 Hz; Zakerana dhaka sp. nov: 2600 – morphological comparisons with the 3800 Hz), and also in the pulse repetition four previously reported species from rate (Chapter III). Principal component Bangladesh as well as other congeners. Z. (PC) and discriminant (DF) analyses of asmati (Fig. 6B) differs from its morphological traits revealed that congeners by several diagnostic Zakerana dhaka sp. nov. also differed characters. These include: SVL 29.1– from all other congers (Chapter III). 33.4 mm; butterfly-shaped vocal marking present in male; forearm length Another distinct lineage of 70% of hand length; relative length of Fejervarya sensu stricto nested with F. fingers, shortest to longest: 2 < 4 < 1 < 3; orissaensis (Chapter II) was identified nostril much closer to snout tip than eye, from Dhaka, where Zakerana dhaka sp. nostril–snout length 57% of distance nov. was found. Z. pierrei was also found from front of eyes to nostril; nostril– from the same locally as a sympatrically

occurring species. This new species from (Poynton and Broadley 1985; Dubois Dhaka had a 3% genetic divergence from 1992). F. orissaensis in mitochondrial genes (16S rRNA and 12S rRNA). Fejervarya Euphlyctis cyanophlyctis was burigangaensis sp. nov. from Dhaka has described from “India orientali" significant morphological differences, [probably from Tranquebar, India supported by PCA and DFA (Chapter II) according to Bauer (1998)] and reported from the series of paratype samples of F. to occur widely in different areas of the orissaensis from Orissa in India. Indian subcontinent (e.g. Dutta and Manamendra-Arachchi 1996; Chanda 2002; Khan 2002; Ao et al. 2003). Even E. Euphlyctis ehrenbergii from Arabian Peninsula was treated as a junior synonym of E. In the mid-seventeenth century, cyanophlyctis until replacement by Fitzinger (1843) described the genus Boulenger (1896, 1920). Several Euphlyctis by designating a type species morphological varieties and sub-species Rana leschenaultii Duméril and Bibron, have been recognized under this species 1841. Later it became a junior synonym (De Silva 1958; Khan 1997b). For to Rana cyanophlyctis Schneider, 1799 example, De Silva (1958) described two (Peters 1863; Günther 1864). Following color varieties from two different areas this, Rana ehrenbergii Peters, 1863, and in Sri Lanka: E. cyanophlyctis (“Rana Rana hexadactylus Lesson, 1834 were cyanophlyctis variety fulvus”) with a identified to belong to this genus yellowish body and “Rana cyanophlictis variety flavens” with a greenish body.

Fig. 7. Phylogenetic relationships among species of the Euphlyctis genus. Analysis based on 746 bp mtDNA (16S and 12S gene) sequence showing the position of Euphlyctis kalasgramensis sp. nov. Numbers on branches refer to bootstrap support and posterior probability from Maximum-likelihood and Bayesian analyses, respectively. The units on the scale indicate branch lengths measured in the number of substitutions per site (next to the branches).

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Furthermore, Khan (1997b) found a where the samples were collected). population from Pakistan with Morphologically, the new species is microscopic spinules scattered on the diagnosable by the following characters body, named E. cyanophlyctis (Chapter IV): snout-vent length (SVL) microspinulata. 30.44 – 37.88 mm, absence of mid-dorsal line, nostril–snout length 3% of SVL, nostril much closer to snout tip than eye, Through the use of molecular nostril–snout length 48% of distance phylogenic analyses, Joshy et al. (2009) from front of eyes to nostril, relative found a new species, E. mudigere near length of fingers (shortest to longest: 1 = the type locality of E. cyanophlyctis. This 2 < 4 < 3), tibia length 59% of SVL, foot new species was earlier confused with E. length 55% of SVL. On the basis of the cyanophlyctis because of their close morphological differences presented in phenotypic resemblance. Similarly, Alam Chapter IV, it is no longer justified to et al. (2008) suggested the possible consider E. kalasgramensis as a occurrence of several cryptic species in morphologically cryptic species within the E. cyanophlyctis species complex on the E. cyanophlyctis species complex. the basis of high genetic divergence of With identification of this new species, mitochondrial gene sequences from the there are currently seven species in this E. cyanophlyctis described from the type genus: E. aloysii, E. cyanophlyctis, E. locality in Southern India near Sri Lanka. ehrenbergii, E. ghoshi, E. hexadactylus, E. kalasgramensis, and E. mudigere (Frost 2016). In my study, I sequenced samples from the Southernmost district of Bangladesh for 16S rRNA and 12S rRNA Euphlyctis kalasgramensis was mitochondrial genes, and found these to earlier considered as E. cyanophlyctis in be cryptic lineages in the genus Bangladesh. This new species has a wide Euphlyctis (Chapter IV), which had been distribution in Bangladesh, as many assigned to the E. cyanophlyctis complex sequences reported by both Alam et al. because of their morphological (2008) and Hasan et al. (2012a) from resemblance to it. This group is highly various regions of Bangladesh match divergent from the samples from the well with the mitochondrial gene presumed type locality of E. sequences of E. kalasgramensis. cyanophlyctis on the basis of sequence divergence (5.5% to 17.8%) in mitochondrial DNA gene sequences. Microhyla Phylogenetic trees constructed with For many years, Microhyla ornata was both Maximum likelihood and Bayesian presumed as one of the most common methods strongly indicated that my Microhyla species in Bangladesh (Kabir samples are a well-supported sister et al. 2009), exhibiting a high degree of taxon to E. mudigere (E. cyanophlyctis + morphological similarity with other E. ehrenbergii) (Chapter IV, Fig. 7). For species in the genus. Several new the application of new nomenclature for candidate species—formerly this taxon, I morphologically diagnosed documented as M. ornata—have been this species from its congeners, and recently reported from Bangladesh named it as “Euphlyctis kalasgramensis (Matsui et al. 2005; Hasan et al. 2012a) sp. nov.”, derived from the locality name based on genetic information and (Kalasgram, a village of Barisal District consideration of the original type locality

identified the specimens from Nilphamari District as being clearly differentiated from all known species in the genus Microhyla, both by detailed morphological comparisons (Fig. 8) and by genetic methods. This Microhyla species from Nilphamari District was formally named as Microhyla nilphamariensis sp. nov. (Chapter V).

Microhyla nilphamariensis sp. nov. is highly divergent (from 5.7% to 13.2% in sequence divergence for 16S rRNA) from other congeneric species. It formed a separate clade in the phylogenetic analyses with high bootstrap (77%) and posterior probability support (0.75), and this new species was recognized as a sister taxon to M. ornata (Chapter V). Phylogenetic analysis found that Microhyla nilphamariensis sp. nov. is nested within the Indian clade of the Microhyla species Fig. 8. Results of the multivariate analyses of group (e.g. M. ornata and M. rubra), morphometric variability in Microhyla nilphamariensis sp. nov., M. ornata and M. rubra. (A) Discriminant and (B) rather than having affinity to Southeast principal component analysis of morphological traits Asian species. Divergence time analyses (Chapter V). showed that the new species diverged from M. ornata about 11.85 mya (5.25 to of M. ornata, which is the Western Ghats 22.46 mya). Molecular clock analyses of India (type locality = “Malabar”, indicated that the South Asian Kerala, India; Duméril and Bibron 1835; Microhylids diverged from the other Biju 2001; Matsui et al. 2005). In this congeneric species about 23 mya ago, thesis (Chapter V), I examined several which corresponds well with the samples collected from Saidpur of geological information on the first Nilphamari District in Bangladesh, which contact between Southeast Asia and were phenotypically similar to M. ornata. India/Bengal basin in the early Miocene To diagnose the collected samples from (22 mya; Alam et al. 2003). their known congeners of Microhyla proved very difficult based on field-level identifications. This is a common Potential threats problem in this genus, because of the high likelihood of homoplasy (Emerson Several factors have been identified as 1986; Bossuyt and Milinkovitch 2000), threats to amphibian species and and also for their minute body size populations, including habitat loss and (Kuramoto and Joshy 2006; Hasan et al. degradation, introduction of alien 2014b). However, genetic comparisons species, emerging pathogens, climate often strongly facilitate differentiation of change, UV-B radiation, pollution, as well existing species and identification of new as direct human-caused mortality candidate species. In Chapter V, I (Stuart et al. 2004; Beebee and Griffiths

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is a fact that the habitats from where the new species were described are habitats experiencing significant threats to amphibian populations (Fujioka and Lane 1997; Lehtinen et al. 1999; Hamer and McDonnell 2008).

In Chapter I, Z. asmati was collected from a 1 km long sewage drainage on Chittagong University campus, filled with mud. From this drainage, 14 amphibian species have been reported thus far (Rasel et al. 2007). In Chapters II and III, both Zakerana dhaka sp. nov. and Fejervarya burigangaensis sp. nov. were found breeding in temporary water pools in the heart of the capital city of Bangladesh. The urban development is a significant threat to biodiversity (e.g. Seto et al. 2012; Newbold et al. 2015), including amphibians (e.g. Gibbs et al. 2005; White 1995). Whether these two species have broader distribution outside of the urban core of Dhaka remains to be investigated, but the fact remains that type localities can be Fig. 9. Maximum-likelihood phylogenetic tree based on variation in considered to be highly vulnerable 16S gene fragment showing the position of Microhyla nilphamariensis sp. nov. in relation to other available Microhyla haplotypes from to expiration. GenBank (Chapter V). Type specimens of 2005; Stuart and Edicions 2008). Apart Euphlyctis kalasgramensis sp. nov. were from the fact that Bangladesh has been collected from an undisturbed pond in among the top frog-leg exporting Kalasgram of Barisal District, countries (Niekisch 1986), the actual Bangladesh (Chapter IV). However, threats to amphibian species and because this species is very common all populations still go undocumented. The over the country – especially in paddy new species described in this thesis were fields in rural areas – it is commonly used all discovered and collected from as live bait for fishing. In 2008, I found disturbed urban habitats or crop fields more than one hundred dead E. within or next to human settlements kalasgramensis individuals from a paddy (Fig. 2; Chapters I, II, III, IV and V). field in Durgapur of Netrakona District, Although more extensive sampling may presumably due to the use of toxic reveal that the described species can also herbicides. The negative effects of be found from more pristine habitats, it agricultural chemicals on amphibians is

a major worldwide concern (e.g. Sparling possible to discover new amphibian et al. 2001; Mann et al. 2009; Van Meter species from highly urbanized areas like et al. 2014). Dhaka. Zakerana dhaka sp. nov. was found in sympatry with Z. pierrei and Fejervarya burigangaensis sp. nov.. However, Zakerana dhaka sp. nov. was CONCLUSIONS AND FUTURE phylogenetically and morphologically DIRECTIONS more closely related to Z. asmati occurring in an Indo-Burmese hilly range The extinction risk of a species is (see Distribution section in Chapter III inversely proportional to its abundance for further details). This disjunct and distribution: abundant and wide- geographical distribution of two closely spread species have lower extinction related species may indicate a history of risk than rare and locally distributed allopatric isolation during the historic species (Johnson 1998). In order to have geological isolation between Indo- a proper assessment of species diversity Burmese and Indian plates. Future study and abundance, correct identification of based on more comprehensive sampling new species and cryptic species is throughout these regions will help to absolutely essential. The incorrect resolve this speculation, as well as identification of ‘cryptic taxa’ as a provide insights to where South Asian common and widely distributed taxa can species meet with the Southeast Asian lead to the wrong assessment of species. Similarly, it was long believed conservation priorities for the species that Euphlyctis cyanophlyctis and (e.g. Nair et al. 2012). In this thesis, I Microhyla ornata are very common and investigated species of several frog widely distributed from the Western genera occurring in South Asia, and Ghats to Bangladesh. In this thesis identified cryptic lineages from their (Chapters IV and V), I found two cryptic congeners. In the beginning of the thesis, lineages which are highly genetically and Zakerana asmati was found as a new morphologically divergent from E. species in Bangladesh: this was the first cyanophlyctis and M. ornata. I formally time in 150 years that a new amphibian named these as Euphlyctis kalasgramesis species has been described from this sp. nov. and Microhyla nilphamariensis country (Theobald 1868). This finding sp. nov.. provided motivation for the rest of this dissertation, and highlighted the In Chapter V, I utilized sequences possibility of finding more undescribed of Microhyla ornata from the GenBank species in other genera. In Chapters II repository, but the collection places for and III, I found two sympatric species many of these sequences were not from two different genera in the same specified, and hence, unknown. After breeding habitats in an urban core of aligning all the GenBank sequences Dhaka city. Zakerana dhaka sp. nov. had allocated to M. ornata, phylogenetic the closest morphological resemblance analysis revealed that many formed a to Z. asmati, and both species were also monophyletic clade with Southeast found to be nested together in the Asian Microhyla species, but some were molecular phylogeny. Bioacoustic very divergent from M. ornata from the analysis approaches were integrated in type locality, and possibly represent Chapter III, allowing comparison of both some yet unrecognized cryptic species Zakerana species with each other. (Fig. 9). Remarkably, some of the Chapters II and III show that it is still sequences designated to M. ornata had

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99% sequence identity to the M. different species. In general, the results nilphamariensis sequences, reported presented in this thesis should prove from the Western Ghats of India [“Bajipe, useful for other amphibian researchers Karnoor, Talagini” as reported in Hasan interested in history and systematics of et al. (2014a)]. Hence, my findings Asian amphibians, as well as for suggest that the distribution of M. conservation biologists interested in nilphamariensis might extend to the amphibians of Southern Asia. Western Ghats of India (Chapter V). Both E. cyanophlyctis and M. ornata are still considered as least concern (LC) by ACKNOWLEDGEMENTS IUCN because of their large distribution ranges. However my findings suggest At first, I would like to express my that reassessment of the actual sincere appreciation and gratitude to my distribution ranges and threats to these supervisor, Prof. Juha Merilä for species is required. In general, by providing me with the opportunity to identifying new taxa this thesis has complete my PhD research project in the provided essential background University of Helsinki. He guided me to information for systematic and my ambition all though my course work, conservation planning of amphibians in inspired me, advised me, and above all, Bangladesh. believed in me that I would make this thesis possible. Molecular phylogenies used in this thesis were based on mitochondrial I also want to thank my co- genes (Chapters I, II, III, IV and V); supervisor Abhilash Nair for his several nuclear genes were included in guidance and helpful suggestions and Chapter II. However, large-scale constant support in all of my papers genomic data would provide a more presented in this thesis. From the solid background for species diversity beginning, he has given me many vital studies and in resolving taxonomic hours for consulting and clarifying affinities. Adding hundreds or thousands science where I had obstacles. of loci could lead to changes in the current phylogenetic hypotheses or even I would like to thank my graduate generic and familial relationships (see thesis committee members Prof. Jyrki review in: Pyron 2015) that I have Muona and Curator Péter Poczai for their presented in this thesis. New genomic helpful advices throughout my time as a approaches can help in resolving PhD student. uncertainties pertaining to generic statuses of many species (e.g. Fejervarya, I would like to thank the thesis pre- Zakerana and Minervarya). Large-scale examiners for their helpful suggestions regional sampling from different which improved the thesis. Especially populations would be helpful in Senior Curator Risto Väinölä provided identifying probable morphological many constructive suggestions that variation, which may further influence greatly improved the scholarship of this diagnostic phenotypic traits used in thesis (possible remaining errors are on taxonomic descriptions. Such sampling my responsibility) would also help in specifying the I am grateful to all of my geographic distributions of the colleagues working in EGRU for their described species, as well as identifying generosity and cooperation, which the environmental threats faced by created a very friendly environment for

me to work in Helsinki University. I I would like to thank my family found myself very lucky to have some members (specially my elder brother, very helpful and friendly persons (Chris sister and mother) for their continuous Eberlein, Nurul Izza Ab Ghani, Jacquelin support in every step of my life, and for DeFaveri, Sergey Morozov, Mehedi inspiring me so that I can achieve my Hasan and Heini Natri) in my office room goal. Rima was always in there over from the 1st day in Helsinki University. phone to remind me for any class and Jacquelin DeFaveri is one of them, she schedule during my PhD studies. has always been very helpful and cooperative whenever any help is I also want to acknowledge all of wanted, specially her language check my friends and field assistants for their over this thesis and manuscripts really support during the fieldwork, specially helped. my friends Ahsan, Tapu, and Pitu Bhai.

I would like to thank all the EGRU Finally, I am dedicating my thesis supportive staffs. Kirsi Kähkönen and to all those frogs, which were sacrificed Sami Karja were very cooperative and during my PhD for the benefit of science. supportive during my lab work. I cannot forget Marika Hölttä, her cooperation REFERENCES helped me to adapt and understand a new life condition in Helsinki. Alam M, Alam MM, Curray JR, et al (2003) An overview of the sedimentary I am also grateful to my divisional geology of the Bengal Basin in (Ecology and Evolution Biology) head relation to the regional tectonic Prof. Veijo Kaitala for his generosity and framework and basin-fill history. kindness to solve my academic and Sedimentary Geology 155:179– administrative difficulties. Similarly 208. LUOVA coordinator Anni Tonteri was Alam MS, Igawa T, Khan MMR, et al very much helpful by providing me with (2008) Genetic divergence and information and answering all of my evolutionary relationships in six queries related to my PhD study. species of genera Hoplobatrachus and Euphlyctis (Amphibia: Anura) This PhD would not be possible from Bangladesh and other Asian without the financial support from the countries revealed by Finnish Cultural Foundation and CIMO, mitochondrial gene sequences. as well as my faculty provided thesis Molecular Phylogenetics and writing grant to me. Evolution 48:515–527. Alho JS, Herczeg G, Laugen AT, et al I would also like to thank the (2011) Allen’s rule revisited: Forest Department, Government of the quantitative genetics of extremity People's Republic of Bangladesh for length in the common frog along a appropriate permissions and local latitudinal gradient. Journal of logistic supports to conduct this Evolutionary Biology 24:59–70. research. Furthermore, I would like to Alroy J (2015) Current extinction rates of thank both Zoological Survey of India reptiles and amphibians. and Zoology Department of the Proceedings of the National University of Chittagong for providing Academy of Sciences 112:13003– access to voucher specimens. 13008.

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