Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften
(Dr. rer. nat.) an der
Universität Konstanz, Mathematisch- Naturwissenschaftliche Sektion
Fachbereich Biologie vorgelegt von
Arie van der Meijden, M.Sc.
Konstanz, März 2006
Prüfungskommission:
Prof. Iwona Adamska Prof. Axel Meyer Prof. Miguel Vences
..love for all living creatures [is] the most noble attribute of man..
Charles Darwin (1809-1882), in “The Descent of Man and Selection in Relation to Sex” (1872).
Acknowledgements
First and foremost I would like to thank Prof. Axel Meyer for hiring me as a technician, and for subsequently showing his confidence in me by allowing me to start my PhD work in his lab. Working in his lab, I have acquired knowledge and skills indispensable to further pursue a career at the high level of biology that Prof. Meyer so adamantly exemplifies, and for this I am very grateful.
No less essential to the realization of this thesis was the guidance of Prof. Miguel Vences, who installed in me a purposeful mode of research, and who showed me the field of amphibian biology as more vibrant, dynamic and enticing as I could have thought.
I am greatly indebted to my collaborators, without whom much of my research would not have been possible, or the results greatly diminished. Apart from Axel Meyer and Miguel Vences, I am therefore thankful to (alphabetical oder) Renaud Boistel, Allan Channing, Ylenia Chiari, Justin Gerlach, Simone Hoegg, Annemarie Ohler, Meike Thomas and David Vieites.
Since my first arrival in Konstanz on October 15th 2002, I have had the privilege of learning lab and analysis techniques from the skilled people who make up the Meyer-lab.
In particular I would like to mention Simone Hoegg, Dirk Steinke and Elke Hespeler for their patience with my initial ignorance.
Outside the realm directly related to my work, I have many people to thank for making the time I spent in Konstanz a lot of fun. The people of the Meyer-lab form a tightly knit community with whom I greatly enjoyed talking and partying with. I would like to thank Ylenia Chiari here in particular, for discussions of work and many other things, and for weathering all the fun and not-so-fun times together with me.
This thesis is a direct result of my incurable affinity to biology, and herpetology in particular, since an early age. Many people have suffered at my hands because of this and I would like to take this space to thank them:
Most of all, I could not have achieved this without the unwavering and full support from my parents. Despite initially poor school results, astronomical electricity bills, lizards in the curtains, snakes in the kitchen, and numerous other escapees, my parents supported
my interests. My sister too patiently endured her share of unexpected encounters around the house. My travels would certainly have never been without my family supporting me.
For the support, understanding and guidance of my family I am therefore especially grateful.
To all my friends who, despite my traveling, kept close contact and who made my every visit to the Netherlands a joy, and who showed or feigned interest all those times I told them something “interesting” about animals, I am very grateful. Special thanks to Lennart Pors for printing and binding this thesis.
I would like to thank Arendo Flipse for putting a steady stream of herpetological biodiversity into my very hands for over twelve years, and for teaching me a lot about herpetology.
To all these people and many more I am greatly indebted, and to all I dedicate this thesis.
Table of Contents
General Introduction ___________________________________________________ 7 Amphibian declines____________________________________________________ 7 Continuous discovery __________________________________________________ 8 Phylogenetic theory as a framework for evolutionary discovery _________________ 9 An introduction to frogs in the superfamily Ranoidea ________________________ 10 Biogeography of the Ranoidea __________________________________________ 12 Taxonomic difficulties owing to homoplasious characters_____________________ 13 Structure of this thesis_________________________________________________ 13 1. Novel phylogenetic relationships of the enigmatic brevicipitine and
scaphiophrynine toads as revealed by sequences from the nuclear rag-1 gene____ 16 1.1 Abstract_________________________________________________________ 16 1.2 Introduction______________________________________________________ 17 1.3 Materials and methods _____________________________________________ 18 1.4 Results__________________________________________________________ 19 1.5 Discussion_______________________________________________________ 20 Acknowledgements ___________________________________________________ 23 2. Comparative performance of the 16S rRNA gene in DNA barcoding of
amphibians___________________________________________________________ 25
2.1 Abstract_________________________________________________________ 25 2.2 Introduction______________________________________________________ 26 2.3 Materials and methods _____________________________________________ 27 2.4 Results__________________________________________________________ 29 2.5 Discussion_______________________________________________________ 37 Acknowledgements ___________________________________________________ 41 3. A previously unrecognised radiation of ranid frogs in Southern Africa revealed by nuclear and mitochondrial DNA sequences _____________________________ 43
3.1 Abstract_________________________________________________________ 43 3.2 Introduction______________________________________________________ 44 3.3 Materials and methods _____________________________________________ 47
3.4 Results__________________________________________________________ 51 3.5 Discussion_______________________________________________________ 54 3.6 Conclusion ______________________________________________________ 59 Acknowledgements ___________________________________________________ 60 4. Molecular phylogenetic evidence for paraphyly of the genus Sooglossus, with the description of a new genus of Seychellean frogs ____________________________ 62
4.1 Abstract_________________________________________________________ 62 4.2 Introduction______________________________________________________ 63 4.3 Materials and methods _____________________________________________ 65 4.4 Results__________________________________________________________ 69 4.5 Discussion_______________________________________________________ 72 Acknowledgements ___________________________________________________ 79 5. Nuclear gene phylogeny of narrow-mouthed toads, family Microhylidae, and a discussion of competing hypotheses for their origin _________________________ 81
5.1 Abstract_________________________________________________________ 81 5.2 Introduction______________________________________________________ 81 5.3 Materials and methods _____________________________________________ 84 5.4 Results__________________________________________________________ 88 5.5 Discussion_______________________________________________________ 91 Acknowledgements ___________________________________________________ 98 Summary ____________________________________________________________ 99
Zusammenfassung____________________________________________________ 102
General references ___________________________________________________ 106
Appendix 1__________________________________________________________ 116
Appendix 2__________________________________________________________ 117
Results produced by collaborators ______________________________________ 119
General Introduction
In this thesis I provide the first broad molecular phylogenetic hypotheses for well known ranoid frog groups, in particular the Brevicipitinae (chapter 1), the Ranidae (chapter 3) and the Microhylidae (chapter 5). Also included in this thesis is a phylogenetic study of the enigmatic Sooglossidae, basal neobatrachians endemic to the Seychelles (chapter 4). I discuss the taxonomic and biogeographic consequences of the recovered phylogenies.
Furthermore the relative merit of the genes 16S and CO1 for genetic barcoding of amphibians is discussed (chapter 2).
Amphibian declines
Since the initial identification of worldwide enigmatic amphibian declines at the world congress of herpetology in 1989, more reports on the disappearance of amphibian populations from all parts of the world have been published (Wake, 1991). The global amphibian assessment (GAA) published their findings on this phenomenon in the journal Science (Stuart et al., 2004), showing that a sobering 32.5% of amphibian species are suffering declines. Moreover, although many are declining due to well understood factors such as introduced species (Vredenburg, 2004), overexploitation (Lannoo et al., 1994) and habitat loss, alteration and fragmentation due to human encroachment (Fisher and Shaffer, 1996; Marsh and Pearman, 1997), 48% of the declining populations are doing so due to unknown reasons. Many factors have been proposed as possible causes of these enigmatic declines; climate change (Pounds et al., 1999; Pounds et al., 2006), chemical contaminants (Hayes et al., 2002), UV radiation (Blaustein et al., 2003), infectious diseases (Daszak, 1998) or a combination of these factors. Four families contribute most to the total number of declining species; Bufonidae, Leptodactylidae, Hylidae and Ranidae (figure 1). Most ranoid frogs (Arthroleptidae, Ranidae, Microhylidae among others) suffer very little or not at all from “enigmatic” declines so far, and most of the affected species are suffering from habitat loss and overexploitation (Stuart et al., 2004).
Despite the overwhelming attention that has rightfully been given to the declines of
unknown cause, most species are in decline directly due to human activities and population expansion. There is therefore a pressing need for conservation actions.
Preservation of taxa selected from the whole tree of life, designed to preserve as much evolutionary history as possible, has proven to be unnecessary (Nee and May, 1997), but for phylogenetically biased extinctions like those of the amphibians (Purvis et al., 2000), such a strategy might be necessary. To identify lineages rich in unique evolutionary history for conservation, a phylogenetic theory of the clade to be pruned by extinction is necessary.
Figure 1 Percentages and numbers of rapidly declining species in amphibian families (with at least one rapidly declining species), broken into groups reflecting the dominant cause of rapid decline:
overexploitation; habitat loss; or enigmatic decline. Taken from Stuart et al. (2004).
Continuous discovery
As instances of amphibian decline and possible extinctions are discovered throughout the world, new species are being discovered at the high rate of 25% in the last 11 years
(Köhler et al., 2005). The use of molecular techniques to identify genetically distinct lineages facilitates the distinction of sibling species (e.g. Ohler and Delorme, 2006), and the reassignment of known taxa to different taxonomic units (see chapter 4). Despite this, most recent discoveries are due to newly collected and described specimens (Bossuyt et al., 2004). The discovery of a representative of a new anuran family (Biju and Bossuyt, 2003) and a plethodontid salamander from Korea (Min et al., 2005), a high endemic diversity on Sri Lanka (Meegaskumbura et al., 2002), as well as the recent discovery of at least 20 possible new frog species in New Guinea (unpublished at time of writing) show that amphibian biodiversity is far from being fully described. It also illustrates our lack of knowledge of relatively well-studied groups as amphibians. As awareness of both the high undescribed amphibian biodiversity and the high rate of uncurbed decline grows, the need to describe and chart “what is out there now” seems to become more urgent (Wilson, 2002). A promising technique that will allow the identification of genetically distinct and possibly unknown lineages is DNA barcoding (Hebert and Gregory, 2005). If the previsions of the proponents of this technique come to pass, handheld devices will allow workers with relatively little training in taxonomy and species identification to identify known species, and thus also identify unknown lineages. This will certainly accelerate the discovery of new amphibian species.
Phylogenetic theory as a framework for evolutionary discovery
The description of evolution as descent with modification from a common ancestor (Darwin, 1859), leads to a dichoto- or polytomous unidirectional branching pattern of evolution. Although this is insufficient to describe the evolutionary process of organisms that engage in horizontal transfer of characters, for most multicellular organisms a bi- or polyfurcating phylogeny describes their evolutionary history well. Knowledge of evolutionary relationships is indispensable to the inference of the direction of acquisition and loss of characters in the course of evolution. As molecular characters, which are largely independent from morphology and biology and therefore a vast source of relatively independent characters, were increasingly used for phylogenetic inference surprising patterns of evolution emerged that were hitherto obscured by ambiguity in morphological datasets (e.g. Zardoya et al., 2003; Zardoya and Meyer, 1996). One group
of thus far notoriously instable taxonomy due to ambiguity in morphological datasets are the ranoid frogs, particularly the families Ranidae and Microhylidae (Frost, 2004).
Although some recent work based on molecular datasets has somewhat alleviated this situation for a small subset of taxa (e.g. Roelants et al., 2004; Vences and Glaw, 2001), broader studies like the ones presented in this thesis (chapters 3 and 5) are increasingly necessary to provide a broad-scale phylogenetic theory.
An introduction to frogs in the superfamily Ranoidea
Here follows a brief introduction to the frogs of the superfamily Ranoidea, as their relationships were understood prior to the studies in this thesis. Although a detailed introduction to the issues of taxonomy and biology of the specific groups can be found at the beginning of each chapter, a broad but brief overview is given here. For the only non ranoid neobatrachian family in this thesis, the Sooglossidae, an introduction is given in chapter 4. The Ranoidea are a subclade of the more inclusive monophyletic crown group of frogs, informally known as the Neobatrachia (see figure 2). Other neobatrachian frogs belong to the superfamily Hyloidea, or to several families of uncertain and possibly basal position; the Heleophrynidae, Myobatrachidae, Nasikabatrachidae and Sooglossidae.
Ranoidea are classically defined by the presence of a firmisternal pectoral girdle. The Ranoidea superfamily can be divided into three epifamilies:
1. Ranoidae, containing the Ranidae, Rhacophoridae and Mantellidae
Ranid frogs are “typical” frogs that can be found leaping from the water’s edge anywhere in Europe, and any lay person asked to describe a frog will probably describe a likeness to a typical ranid. They are therefore known as the “True frogs” and are present on every continent except Antarctica. The family Ranidae has many species (773 species, AmphibiaWeb, accessed February 2006) divided into several highly disputed subgroupings (Frost, 2004). The Ranidae have been suggested to be paraphyletic or polyphyletic relative to the Mantellidae and Rhacophoridae (Ford and Cannatella, 1993).
Recent molecular studies have started to elucidate the phylogenetic relationships among the Ranidae (Hoegg et al., 2004a; Roelants et al., 2004), but the taxonomy of this group is still far from truly reflecting the relationships of its constituents.
Figure 2 Taxonomic classification of the Neobatrachian groups relevant to this thesis. Evidence that was produced in this thesis is not incorporated in this figure, showing large unresolved polytomies in the studied groups.
The Rhacophoridae (273 species) are mostly arboreal, with a high diversity in Asia, but also represented in Africa and on Madagascar. The Mantellidae (157 species) are endemic to Madagascar and have radiated into both arboreal and terrestrial forms.
2. Arthroleptoidae, containing Arthroleptidae, Astylosternidae, Hemisotidae and Hyperoliidae.
All members of this epifamily are restricted to sub-Saharan Africa.
Arthroleptids (32 species) and astylosternids (29 species) are both associated with mesic environments. The arthroleptids possess a horizontal pupil and expanded toe pads, which the astylosternids lack. Hemisotids (9 species) are burrowing ant- and termite specialists which care for their tadpoles in an underground chamber before they are led to open water. Many hyperoliids (260 species) are arboreal but some genera are terrestrial. They are often associated with more xeric environments. The leptopelines are traditionally considered to be part of the Hyperoliidae, but new evidence places this group closer to the arthroleptids (Emerson et al., 2000).
3. Microhyloidae, containing only the Microhylidae
The Microhylidae (422 species), or narrow mouthed frogs, are mostly terrestrial or fossorial forms, although arboreality has evolved several times in this family. Several subfamilies have direct development, or non-feeding larvae. The larvae have a suite characters unique among frogs. They are distributed circumtropically.
Biogeography of the Ranoidea
The Ranoidea superfamily is circumglobally distributed. They are represented on every continent except Antarctica. Of the constituent epifamilies, the Arthroleptoidae (Brevicipitidae) are restricted to sub-Saharan Africa, the Microhyloidae are circumtropically distributed, whereas the Ranoidea enjoy an even wider distribution; they are distributed in the holarctic up to the polar circle, and in the southern hemisphere they are only absent from the southern half of South America, the southern part of Australia, and from New Zealand. Ranoids were initially thought to have originated on Gondwana approximately 140 million years ago (Mya) (Duellman and Trueb, 1986) but more recent molecular clock based divergence time estimates place their initial divergence between 99 Mya (San Mauro et al., 2005) and 69 Mya (Vences et al., 2003b). Current knowledge does not allow for the testing of the various biogeographic scenarios put forth (Duellman and Trueb, 1986; Roelants et al., 2004; Savage, 1973), Although these theories stipulate vicariance due to the breakup of Gondwana and terrestrial dispersal, trans-oceanic dispersal of amphibians cannot be ruled out (Vences et al., 2003b). In chapter 3 and chapter 5, novel biogeographic scenarios based on new data are discussed.
Taxonomic difficulties owing to homoplasious characters
The Anura show a highly conserved body plan (Shubin and Jenkins, 1995). Although the initial divergence within the Anura took place approximately 230 Mya (Graur and Martin, 2004), their basic body plan has not been altered much. This in contrast to the Mammalia, which since the end of the Cretaceous, 65 Mya, have diverged widely. If the anuran body plan is highly constrained, then variability in morphology is probably due to functional adaptations. Since members of different amphibian groups occupy similar ecological niches in various parts of the world, it is likely that homoplasy due to convergence in some instances has occurred (e.g. Bossuyt and Milinkovitch, 2000). In all investigated groups within the Ranoidea, the here presented molecular studies show relationships that were thus far not suspected or difficult to corroborate based on morphological datasets. This high incidence of disagreement between the molecular and morphological data suggests that ranoid taxonomy has been confounded by a prevalence of homoplasy in the morphological characters. This has been suggested to repeatedly complicate the elucidation of phylogenetic relationships among the Microhylidae (Wild, 1995; Zweifel, 1986). Homoplasy has also led to the placement of the arthroleptoid/brevicipitid Brevicipitinae with the Microhylidae (see chapter 1), and has obscured ranid relationships (chapter 3). Alternative to homoplasy, the mosaic retention of ancestral characters in different taxa due to a rapid radiation can also confound the use of these characters for inference of relationships. Although the morphological characters involved in the spurious placements were not studied further, in some instances inferences about the evolution of homoplasious characters can be made (chapter 5). A more detailed and basally better resolved phylogenetic hypothesis will be necessary to map more of these misleading characters.
Structure of this thesis
If, in the light of the current declines and extinctions, we wish to conserve evolutionary history as it is contained in currently living lineages, then knowledge of their phylogeny is a first requirement. Also for the study of evolutionary processes, phylogenetic
knowledge is the basic framework. In this thesis I have used molecular phylogenetic techniques to alleviate the current lack of knowledge on the relationships within important crown group frog taxa. I also have contributed to the selection of a suitable marker for “DNA barcoding”, which promises to aid in further discovering thus far unidentified lineages. The chapters are arranged in chronological order of publication or submission.
Chapter one deals with the microhylid subfamily Brevicipitinae. In our study we show that the rag-1 gene unambiguously places this subfamily outside the Microhylidae, where it had been placed based on morphological characters without discussion.
Chapter two shows the superior performance of the 16S gene as opposed to that of CO1 for the novel application of DNA barcoding, at least for amphibians.
Chapter three shows the potential of molecular phylogeny for discovering relationships that may not be identifiable using morphological markers. Based on a molecular dataset containing three nuclear and two mitochondrial genes, we recovered a unexpected clade of South African frogs with a possibly basal position in the Ranoidae epifamily, and discuss the biogeographic consequences.
Chapter four concentrates on an non-ranoid basal neobatrachian family; the Sooglossidae. These enigmatic Seychellean frogs are organized into two genera. We show that the genus Sooglossus is paraphyletic relative to the genus Nesomantis using two nuclear and one mitochondrial gene and we propose an alternative nomenclature to reflect the new evidence.
Chapter five for the first time shows a molecular phylogenetic theory for the circumtropic family Microhylidae. The molecular dataset consisting of four nuclear genes shows novel relationships, and I discuss the taxonomic and biogeographic implications.
Novel phylogenetic relationships of the enigmatic brevicipitine and scaphiophrynine toads as revealed by sequences from the nuclear rag-1 gene
Breviceps acutirostris
1. Novel phylogenetic relationships of the enigmatic brevicipitine and scaphiophrynine toads as revealed by sequences from the nuclear rag-1 gene
Published:
Van der Meijden, A., M. Vences and A. Meyer. 2004. Novel Phylogenetic relationships of the enigmatic brevicipitine and scaphiophrynine toads as revealed by sequences from the nuclear rag-1 gene. Proceeding of the Royal Society B 271: S378-S381.
1.1 Abstract
Due to a general paucity of characters and an apparently general high level of homoplasy, the systematics of frogs have remained disputed. A phylogeny based on the single-copy nuclear rag-1 gene revealed unexpected placements of scaphiophrynine and brevicipitine toads. The former have usually been considered as sister group to all other extant microhylids or are even classified as their own family. Their basal position among microhylids was weakly indicated in our analysis; but they clearly were part of a strongly supported clade composed of representatives from five other microhylid subfamilies. In contrast, the brevicipitines, a group that hitherto was unanimously considered to belong to the Microhylidae, were highly divergent and placed as a sister group to the arthroleptoid clade. These novel phylogenetic placements are best reflected by a classificatory status of the Scaphiophryninae as subfamily of the Microhylidae, whereas the brevicipitines may merit recognition as distinct family. Our findings seem to corroborate a high degree of morphological homoplasy in frogs and suggest that even highly derived morphological states, such as the hydrostatic tongue of microhylids, hemisotids and brevicipitines, may be subject to convergent evolution, parallelism or character reversal.
1.2 Introduction
Due to their pre-Gondwanan age and cosmopolitan distribution, amphibians are a good model system for the study of biogeography (Duellman and Trueb, 1986; Feller and Hedges, 1998). Their tolerance of salt water is limited; although they are capable of transoceanic dispersal (Vences et al., 2003b) their distribution is likely to have been shaped in great part by vicariance (Duellman and Trueb, 1986).
Application of molecular methods to the elucidation of amphibian phylogeny has revealed surprising instances of morphological homoplasy among regional radiations, e.g.
of Madagascan and Indian tree frogs (Bossuyt and Milinkovitch, 2000), or Indian and African burrowing frogs (Biju and Bossuyt, 2003). These taxa belong to the Neobatrachia, a monophyletic group that contains the wide majority of the recent frogs (Feller and Hedges, 1998; Hoegg et al., 2004a).
Despite the renewed interest in the biogeographic and evolutionary history of anurans, one circumtropic neobatrachian family, the Microhylidae, has so far not been studied through molecular phylogenetic analyses. Although single representatives of this family were included in some works (Biju and Bossuyt, 2003; Feller and Hedges, 1998), the intrafamilial relationships remain unstudied from a molecular perspective.
The Microhylidae contains 349 species in 67 genera (excluding scaphiophrynines), occurring in the Americas, sub-Saharan Africa, Madagascar, India and most of southeast Asia to New Guinea and northernmost Australia (AmphibiaWeb.org, as of 2003).
Microhylids are defined by a uniquely derived tadpole morpholog (type II of Orton, 1952), by an osteological trend towards reduction of shoulder girdle elements, and by a specialized microphagous feeding behaviour with hydrostatic tongues (Meyers et al., 2004).
Among microhylids, the phylogenetic position of the eight species in the subfamily Scaphiophryninae from Madagascar is especially enigmatic. Scaphiophrynine tadpoles are intermediate between Orton's tadpole types II and IV (Orton, 1952), the latter being the generalized neobatrachian type (Wassersug, 1984). Scaphiophrynines were placed within the Ranidae until Guibé (1956) placed them into the Microhylidae. Savage (1973) suggested their inclusion in yet another family, the Hyperoliidae. Dubois (Dubois, 1992) raised them to family rank as Scaphiophrynidae. Another microhylid subfamily of
uncertain affinities is the African Brevicipitinae, or rain frogs, composed of 18 species in five genera. Interestingly, these are the only microhylids in which direct development occurs, posing difficulties for an assessment of their larval features.
Here we present the first data on the phylogenetic position of scaphiophrynine and brevicipitine toads using DNA sequences of a single copy nuclear gene, rag-1, which is known to provide an adequate resolution in the analysis of anuran relationships (Hoegg et al., 2004a). Surprisingly our results indicate that brevicipitines might not belong into the microhylid lineage while scaphiophrynines do, contrary to current classification and morphological evidence.
1.3 Materials and methods
Taxa were selected to cover major clades among ranoid neobatrachians to which microhylids are known to belong (Biju and Bossuyt, 2003; Hoegg et al., 2004a). We included taxa of six out of the nine microhylid subfamilies accepted by Duellman &
Trueb (1986), i.e., all except the Asterophryinae, Genyophryninae and Melanobatrachinae. The archaeobatrachian Xenopus, and several hyloid neobatrachians, were used as hierarchical outgroups. A list of taxa and GenBank accession numbers are given in table 1.1.
DNA was extracted from muscle tissue stored at -80°C or fixed in 70% ethanol. Tissue samples were digested using Proteinase K (final concentration 1 mg/mL), homogenised and subsequently purified following a standard salt extraction protocol. We used primers as in Hoegg et al. (2004). PCR was performed in 25 µL reactions containing 0.5-1.0 units of REDTaq DNA Polymerase (Sigma, Taufkirchen, Germany), 0.01 units of Pwo DNA polymerase (Roche, Mannheim, Germany), 50 ng genomic DNA, 10 pmol of each primer, 15 nmol of each dNTP, 50 nmol additional MgCl2 and the REDTaq PCR reaction buffer (one fold concentrated: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.1 mM MgCl2 and 0.01% gelatine). Cycle conditions were adapted from a long range PCR protocol (Barnes, 1994), with an initial denaturation step at 94°C for 5 minutes, followed by ten cycles with 94°C for 30 seconds, annealing temperatures increasing by 0.5°C per cycle from 52 to 57°C and extending for 3 minutes at 68°C. Additional 20 cycles were performed with
94°C for 10 seconds, 57°C for 40 seconds and 68°C for 3 minutes. The final extension was done at 68°C for 5 minutes.
PCR products were purified directly via spin columns (QIAGEN). Sequencing was performed directly using the corresponding PCR primers (forward and reverse).
DNA sequences of both strands were obtained using the BigDye Terminator cycle- sequencing ready reaction kit (Applied Biosystems Inc.) on an ABI 3100 capillary sequencer using the manufacturer’s instructions.
Maximum Parsimony (MP) and Maximum Likelihood (ML) phylogenies were calculated using PAUP* (Swofford, 2002). The best fitting models of sequence evolution for ML analyses were obtained by Modeltest 3.06 (Posada and Crandall, 1998). Heuristic searches were performed using 10 replicates of a stepwise addition of taxa.
Robustness of the MP tree topology was tested by bootstrap analysis with 2000 replicates; 500 ML bootstrap replicates were calculated. Bayesian Inference was conducted with MrBayes 2.0 (Huelsenbeck and Ronquist, 2001) using the GTR model with 100,000 generations, sampling trees every 10th generation, (and calculating a consensus tree after omitting the first 5,000 trees) ("burn-in" set at 1000).
We tested alternative phylogenetic hypotheses using Shimodaira-Hasegawa tests as implemented in PAUP*, with RELL optimization and 1000 bootstrap replicates. To avoid biases by the previous selection of alternative topologies we applied the SH test simultaneously to all possible unrooted trees in a reduced set of six taxa, containing Breviceps, Scaphiophryne, Bufo regularis as outgroup, and the microhylid, arthroleptoid and ranoid taxa with the shortest branch length each (Plethodontohyla, Kassina, Lankanectes), assuming that short branch lengths indicate a low number of autapomorphies that could mask phylogenetic affinities
1.4 Results
The dataset consisted of 1566 DNA positions in 33 species. The trees obtained through MP, ML and Bayesian methods (figure 1.1) subdivide the ranoids into three well supported major clades, corresponding to the epifamilies Ranoidae, Microhyloidae and the Arthroleptoidae as defined by Vences and Glaw (2001). The Ranoidae contained the families Rhacophoridae and Mantellidae, and the paraphyletic Ranidae. Within the
Arthroleptoidae, the hyperoliid Leptopelis is a sister taxon of Arthroleptis, rendering Hyperoliidae paraphyletic with respect to Arthroleptis.
Microhylids formed a highly supported clade that contained Scaphiophryne but not Breviceps. Within this clade, relationships were poorly resolved due to very short basal branch lengths, suggesting the possibility of a rapid lineage formation early in the evolution of this group. The ML and Bayesian analyses placed Scaphiophryne as a sister group to the remaining microhylids. However this placement did not receive strong support, and the differentiation of Scaphiophryne within the clade of the remaining microhylids was small as indicated by the short branch lengths between splits in this clade. Breviceps did not group with other microhylids but instead was the sister group of the Arthroleptoidae.
All alternative tree topologies reflecting current classification, i.e., Breviceps is part of the Microhylidae while Scaphiophryne is not, were significantly rejected by the SH tests:
Plethodontohyla was the sister group of Scaphiophryne and not of Breviceps in the reduced set of taxa analysed (P<0.001). However, maintaining a sister-group relationship of Scaphiophryne and Plethodontohyla, alternative positions of Breviceps could not be significantly excluded; this applied also to its placement as the sister group to the (Scaphiophryne, Plethodontohyla) clade.
1.5 Discussion
Larvae of Scaphiophryne are characterized by morphological characters that are considered to be plesiomorphic relative to the highly specialized, suspension-feeding microhylid type (Haas, 2003; Wassersug, 1984). Their derived larval traits define microhylids as monophyletic group to the exclusion of Scaphiophryne. The most parsimonious phylogeny based on this character complex therefore would expect this genus to occupy a distinctly basal position relative to other microhylids. However, what seems clear from the tree shape (figure 1.1) is that scaphiophrynines did not diverge particularly early in microhylid evolution but were one of the major lineages in the initial radiation of these frogs. This indicates a fast evolutionary transition from the Scaphiophryne-like tadpole morphology to a derived microhylid tadpole.
Figure 1.1 Maximum Likelihood tree based on the analysis of 1566 base pairs of the rag-1 gene, highlighting the phylogenetic position of scaphiophrynines (Scaphiophryne) and brevicipitines (Breviceps) among ranoid neobatrachians. The numbers indicate on the branches are bootstrap support values in percent of ML (100 replicates) and MP (2000 replicates) searches. Asterisks placed to the right of nodes indicate Bayesian posterior probabilities >95%. The tree was rooted with Xenopus laevis (not shown). The insert pictures show representatives of the genera Scaphiophryne and Breviceps .
Also, the tree presented by Biju and Bossuyt (2003) was unambiguous in suggesting a sister-group relationship between scaphiophrynines and other microhylids. From a classificatory point of view, this phylogenetic pattern would best be reflected by a status as subfamily of the Microhylidae rather than as separate family. The family Microhylidae, according to our analysis, contains endemic genera from South America (Dermatonotus), Asia (Kaloula), Africa (Phrynomantis) and Madagascar (Dyscophus, Scaphiophryne, Plethodontohyla). The Madagascan taxa were not a monophyletic clade.
Instead, Dyscophus grouped with the Asian Kaloula, indicating possible intercontinental relationships parallel to those of the rhacophorid (Asia) and mantellid (Madagascar) tree frogs (Biju and Bossuyt, 2003; Bossuyt and Milinkovitch, 2000).
Surprisingly, the African Breviceps (Brevicipitinae) was resolved not as being part of the microhylid clade, but they were grouped with the Arthroleptoidae. This placement differs from conclusions based on morphological and mitochondrial characters (e.g., Emerson et al., 2000). Our data were not sufficient to significantly exclude alternative phylogenetic hypotheses, but the SH tests did significantly exclude the classical hypothesis, in which the brevicipitines are part of the Microhylidae to the exclusion of Scaphiophryne. If confirmed by further data sets, the grouping favoured by our analysis would suggest that the specialized hydrostatic tongue that is characteristic for microhylids including brevicipitines (Meyers et al., 2004) was reversed back to a more generalized state in the Arthroleptoidae, or, possibly even more interestingly, evolved convergently or in parallel at least twice (in brevicipitines and in microhylids). This hypothesis is further supported by the finding that Hemisus, another taxon characterized by a hydrostatic tongue, groups with arthroleptoids rather than with microhylids as usually thought (Biju and Bossuyt, 2003). The separate phylogenetic placement of brevicipitines from other microhylids, together with the possession of several striking morphological specialisations shared only with the Microhylidae and Hemisotidae, might justify a change in their classificatory assignment, i.e., their inclusion in an own family.
Microhylids are characterized by a high variability in their osteological characters due to the repeated evolution of fossoriality and the effects of miniaturization (Wild, 1995).
Osteological characters are usually more conservative in anurans and are therefore considered to be informative features for higher level taxonomy (Duellman and Trueb,
1986). The unexpected molecular phylogenetic placement of scaphiophrynine and brevicipitine toads, if further confirmed, could provide a stark example for the high level of homoplasy in morphological characters in anurans and indicates that, in these organisms, convergent evolution and reversals may be possible even in seemingly highly derived morphological traits.
Acknowledgements
We are grateful to Marius Burger, Alan Channing, Frank Glaw and Stefan Wanke for their help during sample collection and to Simone Hoegg and Dirk Steinke for valuable comments and technical assistance. We thank three anonymous reviewers for their helpful comments on the manuscript. Financial support was provided through grants of the Deutsche Forschungsgemeinschaft to M.V. and A.M.
Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians
2. Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians
Published: Vences, M., M. Thomas, A. van der Meijden, Y. Chiari, D. R. Vieites. 2005.
Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians.
Frontiers in Zoology 2: article 5
2.1 Abstract
Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI) has been proposed as universal marker for this purpose among animals. Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combinations of COI primers had lower success rates. COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1- 17%), with low degrees of pairwise haplotype divergence within populations (0-1%). We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.
2.2 Introduction
The use of short DNA sequences for the standardized identification of organisms has recently gained attention under the terms DNA barcoding or DNA taxonomy (Floyd et al., 2002; Hebert et al., 2003b; Tautz et al., 2003). Among the promising applications of this method are the assignments of unknown life-history stages to adult organisms (Hebert et al., 2004c; Thomas et al., 2005), the large-scale identification of organisms in ecological or genomic studies (Blaxter, 2004; Floyd et al., 2002) and, most controversially, explorative studies to discover potentially undescribed "candidate"
species (Hebert et al., 2004c; Hebert et al., 2004d; Venter et al., 2004). Although it is not a fundamentally new technique (Moritz and Cicero, 2004), DNA barcoding is promising because technical progress has made its large-scale, automated application feasible (Blaxter, 2004; Tautz et al., 2003), which may accelerate taxonomic progress (Wilson, 2004).
DNA barcoding and DNA taxonomy are realities in many fields (Blaxter, 2004).
Consensus by the scientific community is essential with respect to the most suitable genes that allow robust and repeatable amplification and sequencing and provide unequivocal resolution to identify a broad spectrum of organisms. While D. Tautz and co-workers (Tautz et al., 2003) proposed the nuclear ribosomal RNA genes for this purpose, P. D. N. Hebert and colleagues have strongly argued in favor of a 5' fragment of the mitochondrial gene for cytochrome oxidase, subunit I (COI or COXI) (Hebert et al., 2003b; Hebert et al., 2003c). This gene fragment has been shown to provide a sufficient resolution and robustness in some groups of organisms, such as arthropods and, more recently, birds (Hebert et al., 2003b; Hebert et al., 2004c; Hebert et al., 2003c; Hebert et al., 2004d).
A genetic marker suitable for DNA barcoding needs to meet a number of criteria (Hebert et al., 2003a). First, in the study group, it needs to be sufficiently variable to be able to discriminate among most species but sufficiently conserved to be less variable within than between species. Second, priming sites need to be sufficiently conserved to permit a reliable amplification without the risk of false negatives when pooled samples or environmental DNA is analyzed. Third, the gene should convey sufficient phylogenetic information to assign species to major taxa using simple phenetic approaches. Fourth, its
amplification and sequencing should be as robust as possible, also under variable lab conditions and protocols. Fifth, sequence alignment should be possible also among distantly related taxa.
Here we explore the performance of a fragment of the 16S ribosomal RNA gene (16S) in DNA barcoding of amphibians. As a contribution to the discussion about suitable standard markers we provide experimental data on comparative amplification success of 16S and COI in amphibians, empirical data on conservedness of priming sites, and an example from the 16S-based identification of amphibian larval stages.
2.3 Materials and methods
To test for universality of primers and cycling conditions, we performed parallel experiments in three different laboratories (Berkeley, Cologne, Konstanz) using the same primers but different biochemical products and thermocyclers, and slightly different protocols.
The selected primers for 16S (Palumbi et al., 1991) amplify a fragment of ca. 550 bp (in amphibians) that has been used in many phylogenetic and phylogeographic studies in this and other vertebrate classes: 16SA-L, 5' - CGC CTG TTT ATC AAA AAC AT - 3';
16SB-H, 5' - CCG GTC TGA ACT CAG ATC ACG T - 3'.
For COI we tested (1) three primers designed for birds (Hebert et al., 2004b) that amplify a 749 bp region near the 5'-terminus of this gene: BirdF1, 5' - TTC TCC AAC CAC AAA GAC ATT GGC AC - 3', BirdR1, 5' - ACG TGG GAG ATA ATT CCA AAT CCT G - 3', and BirdR2, 5' - ACT ACA TGT GAG ATG ATT CCG AAT CCA G - 3'; and (2) one pair of primers designed for arthropods (Hebert et al., 2003a) that amplify a 658 bp fragment in the same region: LCO1490, 5' - GGT CAA CAA ATC ATA AAG ATA TTG G - 3', and HCO2198, 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3'.
Sequences of additional primers for COI that had performed well in mammals and fishes were kindly made available by P. D. N. Hebert (personal communication in 2004) and these primers yielded similar results (not shown).
The optimal annealing temperatures for the COI primers were determined using a gradient thermocycler and were found to be 49-50°C; the 16S annealing temperature was 55°C. Successfully amplified fragments were sequenced using various automated
sequencers and deposited in GenBank. Accession numbers for the complete data set of adult mantellid sequences used for the assessment of intra- and interspecific divergences (e.g., figure 2.5) are AY847959-AY848683. Accession numbers of the obtained COI sequences are AY883978-AY883995.
Nucleotide variability was scored using the software DnaSP (Rozas et al., 2003) at COI and 16S priming sites of the following complete mitochondrial genomes of nine amphibians and 59 other vertebrates: Cephalochordata: AF098298, Branchiostoma.
Myxiniformes: AJ404477, Myxine. Petromyzontiformes: U11880, Petromyzon.
Chondrichthyes: AJ310140, Chimaera; AF106038, Raja; Y16067, Scyliorhinus;
Y18134, Squalus. Actinopterygii: AY442347, Amia; AB038556, Anguilla; AB034824, Coregonus; M91245, Crossostoma; AP002944, Gasterosteus; AB047553, Plecoglossus;
U62532, Polypterus; U12143, Salmo. Dipnoi: L42813, Protopterus. Coelacanthiformes:
U82228, Latimeria. Amphibia, Gymnophiona: AF154051, Typhlonectes. Amphibia, Urodela: AJ584639, Ambystoma; AJ492192, Andrias; AF154053, Mertensiella;
AJ419960, Ranodon. Amphibia, Anura: AB127977, Buergeria; NC_005794, Bufo;
AY158705; Fejervarya; AB043889, Rana; M10217, Xenopus. Testudines: AF069423, NC_000886, Chelonia; Chrysemys; AF366350, Dogania; AY687385, Pelodiscus;
AF039066, Pelomedusa. Squamata: NC_005958, Abronia; AB079613, Cordylus;
AB008539, Dinodon; AJ278511, Iguana; AB079597, Leptotyphlops; AB079242, Sceloporus; AB080274, Shinisaurus. Crocodilia: AJ404872, Caiman. Aves: AF363031, Anser; AY074885, Arenaria; AF090337, Aythya; AF380305, Buteo; AB026818, Ciconia;
AF362763, Eudyptula; AF090338, Falco; AY235571, Gallus; AY074886, Haematopus;
AF090339, Rhea; Y12025, Struthio. Mammalia: X83427, Ornithorhynchus; Y10524, Macropus; AJ304826, Vombatus; AF061340, Artibeus; U96639, Canis; AJ222767, Cavia ; AY075116, Dugong; AB099484, Echinops; Y19184, Lama; AJ224821, Loxodonta; AB042432, Mus; AJ001562, Myoxus; AJ001588, Oryctolagus; AF321050, Pteropus; AB061527, Sorex; AF348159, Tarsius; AF217811, Tupaia; AF303111, Ursus (for species names, see GenBank under the respective accession numbers).
16S sequences of a large sample of Madagascan frogs were used to build a database in BioEdit (North Carolina State University). Tadpole sequences were compared with this database using local BLAST searches (Altschul et al., 1990) as implemented in BioEdit.
The performance of COI and 16S in assigning taxa to inclusive major clades was tested based on gene fragments homologous to those amplified by the primers used herein (see above), extracted from the complete mitochondrial sequences of 68 vertebrate taxa.
Sequences were aligned in Sequence Navigator (Applied Biosystems) by a Clustal algorithm with a gap penalty of 50, a gap extend penalty of 10 and a setting of the ktup parameter at 2. PAUP* (Swofford, 2002) was used with the neighbor-joining algorithm and LogDet distances and excluding pairwise comparisons for gapped sites. We chose these simple phenetic methods instead of maximum likelihood or maximum parsimony approaches because they are computationally more demanding and because the aim of DNA barcoding is a robust and fast identification of taxa rather than an accurate determination of their phylogenetic relationships.
2.4 Results
Amplification experiments
We performed independent amplification experiments with one set of 16S primers and three published sets of COI primers (Hebert et al., 2003a; Hebert et al., 2004b) focusing on representatives of different frog, salamander and caecilian genera. The experiments were concordant in yielding more general results for 16S than COI. In a set of of fresh and well-preserved samples from relatively closely related mantellid frogs from Madagascar (appendix 1) the 16S amplification success was complete, whereas the three sets of COI primers yielded success rates of only 50-70%. Considering all three primer combinations, there were two species of frogs (10%) that did not amplify for COI (Boophis septentrionalis and B. tephraeomystax) at all.
Priming sites
The variability of priming sites was surveyed using nine complete amphibian mitochondrial sequences from GenBank (figure 2.1), and 59 mt genomes of fishes, reptiles, birds and mammals (figure 2.2).
Figure 2.1 Variability of priming sites for 16S rRNA and COI in amphibians.
Figure 2.2 Variation in priming sites of 16S rRNA (a, F-primer; b, R-primer) and COI (c, Bird-F1, LCO1490; d, HCO2198; e, Bird-R1, Bird-R2) fragments studied herein. Values are nucleotide variability as calculated using the DnaSP program. Grey bars show the values for nine amphibians, black bars the values for a set of 59 other vertebrates (see Materials and methods, and figures 2.3 and 2.4).
A high variability was encountered for COI. The sequences of some species were largely consistent with the primers: Xenopus had two mutations only at each of the priming regions. However, other sequences were strongly different, with up to seven mutations, all at third codon positions. No particular pattern was recognizable for any major group that would facilitate designing COI primers specific for frogs, salamanders or caecilians.
Interestingly the variability among the amphibian sequences available was as large as or larger than among the complete set of vertebrates at many nucleotide positions of COI priming sites (figure 2.2), indicating a possible higher than random variability of this gene in amphibians.
In contrast, the 16S priming sites were remarkably constant both among amphibians and among other vertebrates (figures 2.1 and 2.2). A wider survey of priming sites, i.e., the alternative reverse priming sites used in arthropod and bird studies (Hebert et al., 2003a;
Hebert et al., 2004b) confirmed the high variability of COI in amphibians, and in vertebrates in general (figure 2.2). A screening of the first 800 bp of the C-terminal part of the gene in nine amphibians of which complete mitochondrial genes were available did not reveal a single fragment of 20 bp where all nine species would agree in 80% or more of their nucleotides.
Recovery of major groups
The phenetic neighbor-joining analysis using the 16S fragment produced a tree that contained eight major groupings that conform to or are congruent with the current classification and phylogeny (figure 2.3): cartilaginous fishes, salamanders, frogs, turtles, eutherian mammals, mammals, squamates, birds. Of these, the COI tree (figure 2.4) recovered only the lineages of cartilaginuous fishes and birds. No additional relevant major lineage was found in the COI analysis that had not been recovered also by the 16S analysis.
Figure 2.3 Neighbor-joining tree of selected vertebrate taxa based on the fragment of the 16S rRNA gene amplified by primers 16SaL and 16SbH. Numbers in black circles indicate major clades that were recovered by this analysis: (1) cartilaginous fishes, (2) salamanders, (3) frogs, (4) turtles, (5) eutherian mammals, (6) mammals, (7) squamates, (8) birds.
Figure 2.4 Neighbor-joining tree of selected vertebrate taxa based on the fragment of the COI gene amplified by primers LCO1490 and HCO2198. Numbers in black circles indicate major clades that were recovered by this analysis. Only two of the clades recovered by the 16S analysis are also monophyletic here: (1) cartilaginous fishes, (8) birds.
16S rRNA barcoding of tadpoles
From an ongoing project involving the large-scale identification of tadpoles of Madagascan frogs (Thomas et al., 2005) we here provide data from larval and adult frog
species from two sites of high anuran diversity in eastern Madagascar, Andasibe and Ranomafana. These two localities are separated by a geographical distance of ca. 250 km.
The results will be presented in more detail elsewhere.
We selected target species for which morphological and bioacoustic uniformity suggests that populations from Ranomafana and Andasibe are conspecific. All these species belong to the family Mantellidae. We then analysed haplotypes within and between these populations. In addition we assessed divergences among sibling species of mantellid frogs. These were defined as morphologically similar species that are phylogenetically sister to each other, or are in well-defined but phylogenetically poorly resolved clades of 3-5 species. Results revealed a low intrapopulational variation of 0-3% uncorrected pairwise distances in the 16S gene, a surprisingly large differentiation among conspecific populations of 0-5.1%, and a wide range of differentiation among species, ranging from 1-16.5% with a mode at 7-9% (figure 2.5). The few species separated by low genetic distances were allopatrically distributed. The interspecific divergence was higher in those species pairs in which syntopic occurrence has been recorded or is likely (2.7-16.5%
divergence, mean 8.5%) as compared to those that so far only have been found in allopatry (1.0-12.9%, mean 6.9%).
Phylogenetic and phenetic analyses (Bayesian and Neighbor-joining) of these and many additional sequences (to be published elsewhere) mostly grouped sequences of those specimens from Ranomafana and Andasibe that a priori had been considered to be conspecific (exceptions were Mantidactylus boulengeri, not considered in the intraspecific comparisons here, and M. blommersae). This indicates that cases, in which haplotypes of a species are more similar to those of another species than to those of other conspecific individuals or populations, are rare in these frogs. Sharing of identical haplotypes among individuals belonging to different species, in our dataset, was limited to three closely related species pairs of low genetic divergences: Boophis doulioti and B.
tephraeomystax, B. goudoti and B. cf. periegetes, Mantella aurantiaca and M. crocea.
Depending on the taxonomic scheme employed, our complete data set contains 200-300 species of Madagascan frogs. Hence, haplotype sharing was demonstrated in 2-3% of the total number of species only.
Figure 2.5 Variation in the fragment of the 16S rRNA gene (ca. 550 bp) studied herein, (a) within populations, (b) among conspecific populations and (c) among sibling species of frogs in the family Mantellidae from Madagascar. The values are uncorrected p-distances from pairwise comparisons in the respective category. Only one (mean) value per species was used in (a) and (b), even when multiple individuals were compared.
To explore the reliability of tadpole identification using the 16S gene we used local BLAST searches against a database containing about 1000 sequences of adult frogs from a wide sampling all over Madagascar. 138 tadpoles from the Andasibe region and 84 tadpoles from the Ranomafana region were compared with adult sequences in the database. In 77% of the cases the highest scores were those from comparisons to adults from the same site as the tadpoles. In most of the unsuccessful comparisons, adult sequences of the corresponding species were not available from the tadpole site (21%). In
only 5 cases (2%) conspecific adults collected from a different site than the tadpoles yielded higher BLAST scores although adult sequences from the same site were in the database.
2.5 Discussion
DNA barcoding in amphibians
DNA barcoding has great appeal as a universally applicable tool for identification of species and variants of organisms, possibly even in automated handheld devices (Janzen, 2004). However, doubtless severe restrictions exist to its universal applicability (Moritz and Cicero, 2004). Some taxa, e.g., cichlid fishes of Lake Victoria, have radiated so rapidly that the speciation events have not left any traces in their mitochondrial genomes (Verheyen et al., 2003); identifying these species genetically will only be possible through the examination of multiple nuclear markers, as it has been done to assess their phylogeny (Albertson et al., 1999). Some snails are characterized by a high intraspecific haplotype diversity, which could disable attempts to identify and distinguish among species using such markers (Thomaz et al., 1996).
Haplotype sharing due to incomplete lineage sorting or introgression is also known in amphibians (Funk and Omland, 2003) although it was not common in mantellid frogs in our data set. However, a number of species showed haplotype sharing with other species, or non-monophyletic haplotypes, warranting a more extensive discussion. In Mantidactylus boulengeri, specimens from Andasibe and Ranomafana have similar advertisement calls and (at least superficially) similar morphologies, but their 16S haplotypes were not a monophyletic group (unpublished data). This species belongs to a group of direct-developing frogs that, like the Neotropical Eleutherodactylus (Dubois, 2004) may be characterized by a high rate of cryptic speciation. Further data are necessary to decide whether the populations from Ranomafana and Andasibe are indeed conspecific. In contrast, there is little doubt that the populations of Mantidactylus blommersae from these two sites are conspecific, yet the Ranomafana haplotypes are closer to those of the clearly distinct species M. domerguei. The species pairs where haplotype sharing has been observed (see Results) all appear to be allopatrically to parapatrically distributed and show no or only low differences in advertisement calls,
indicating that occasional hybridization along contact zones may be possible (e.g., Chiari et al., 2004). Haplotypes of each of these species pairs always formed highly supported clusters or clades, and had divergences below 3%, indicating that haplotype sharing in mantellids may only constitute a problem when individuals are to be assigned to such closely related sister species.
Although our data show that DNA barcoding in mantellids is a largely valid approach when both reference and test sequences come from the same site, the occurrence of non- monophyletic and highly divergent haplotypes within species characterizes these and other amphibians as a challenging group for this technique. Certainly, DNA barcoding is unable to provide a fully reliable species identification in amphibians, especially if reference sequences do not cover the entire genetic variability and geographic distribution of a species. However, the same is true for any other morphological or bioacoustic identification method. Case studies are needed to estimate more precisely the margin of error of molecular identification of amphibian species. For many approaches, such as the molecular survey of the trade in frog legs for human consumption (Veith et al., 2000), the error margins might be acceptable. In contrast, the broad overlap of intraspecific and interspecific divergences (figure 2.5) cautions against simplistic diagnoses of presumably new amphibian species by DNA divergences alone. A large proportion of biological and evolutionary species will be missed by inventories that characterize candidate species by DNA divergences above a previously defined threshold.
Comparative performance of DNA barcoding markers in amphibians
Phenomena of haplotype sharing or non-monophyletic conspecific haplotypes will affect any DNA barcoding approach that uses mitochondrial genes, and are also to be expected with nuclear genes (e.g., Machado and Hey, 2003). Nevertheless, some genes certainly outperform others in terms of discriminatory power and universal applicability, and these characteristics may also vary among organism groups. The mitochondria of plants are characterized by very different evolutionary patterns than those of animals, including frequent translocation of genetic material into and from the nucleus (Palmer et al., 2004), which limits their use for DNA barcoding purposes. Nuclear ribosomal DNA (18S and
28S), proposed as standard marker (Tautz et al., 2003), has a high potential in invertebrate DNA barcoding but its high-throughput amplification encounters difficulties in vertebrates.
As a consequence, despite the need of consensus on markers for universal applicability of DNA barcoding, the use of different genes in different groups of organisms seems reasonable. It has been hypothesized that universal COI primers may enable amplification of a 5' terminal fragment from representatives of most animal phyla due to their robustness (Hebert et al., 2003a). The success in DNA barcoding of lepidopterans and birds suggests that this gene fragment can indeed be used as a standard for many higher animal taxa (Hebert et al., 2003a; Hebert et al., 2004a; Hebert et al., 2004b).
In our experiments we compared 16S primers commonly used in amphibians to COI primers that had been developed for other vertebrates (Hebert et al., 2004b) or invertebrates (Hebert et al., 2003a). It may well be possible, with some effort, to design primers that are more successful and consistent in amplifying COI from amphibians.
However, our results from mantellid frogs (appendix 1) indicate a very good amplification success of the primers for some species, but failure for other, related species. This and our results on variability of priming sites predict enormous difficulties in designing one pair of primers that will reliably amplify this gene fragment in all vertebrates, all amphibians, or even all representatives of any amphibian order. A set of one forward and three reverse COI primers have been successfully used to amplify and sequence a large number of bird species (Hebert et al., 2004b), but birds are a much younger clade than amphibians with a probably lower mitochondrial variability.
A further optimization of COI amplification may also be achieved regarding the PCR protocol. Herein we used standard protocols that optimized annealing temperature only, whereas more complex touchdown protocols have been used for birds and butterflies (Hebert et al., 2004a; Hebert et al., 2004b). However, one major requirement for a DNA barcoding marker is its robustness to variable lab conditions. If DNA barcoding is to be applied as a standard in many different labs, primers and genes need to be chosen that amplify reliably under very different conditions and under standard protocols. This clearly applies to 16S, which we have amplified with very different annealing temperatures and PCR conditions in previous exploratory studies (results not shown).
Alignment of 16S sequences is complicated by the prevalence of insertions and deletions, and this gene is less variable than COI (Hebert et al., 2003a). Nevertheless, our results indicate that even using an uncritical automated alignment this gene has a higher potential than COI to assign vertebrate sequences to the level of classes and orders.
The 16S gene is a highly conserved mitochondrial marker but mutations are common in some variable regions, corresponding to loops in the ribosomal RNA structure. In amphibians, where many species are relatively old entities (Wilson et al., 1974), this ensures a sufficient amount of mutations among species. Our results for amphibians, and previous experience with fishes, reptiles and mammals, indicate that 16S is sufficiently variable to unambiguously identify most species.
A further mitochondrial gene that has been widely used in amphibian phylogenetic and phylogeographic studies is cytochrome b. This gene can easily be amplified in salamanders and archaeobatrachian frogs using primers that anneal with adjacent tRNA genes. However, neobatrachian frogs (the wide majority of amphibian species) are characterized by rearrangements of the mitochondrial genome (Macey et al., 1997;
Sumida et al., 2001), and cytochrome b in these species borders directly to the control region. Although cytochrome b primers are available that work in many neobatrachians (Bossuyt and Milinkovitch, 2000; Vences et al., 2003b), they sometimes fail in closely related forms, similar to the COI primers used herein, presumably because of mutations at the priming sites (pers. obs. M. Vences in mantellid frogs).
In contrast, the 16S primer pair used here can be considered as truly universal not only for amphibians but even for vertebrates. This is also reflected by the high number of amphibian 16S sequences in GenBank (2620 hits for 16S vs. 483 hits for COI, as of September 2004). Moreover, the 16S and 12S rRNA genes have been selected as standard markers for phylogeny reconstruction in amphibians (AmphibiaTree consortium), which will lead to a near-complete global dataset of amphibian 16S sequences in the near future.
If the development of handheld devices (Janzen, 2004) is envisaged as a realistic goal, then the universality and robustness of primers should be among the most relevant characteristics of a gene for DNA barcoding. When pooled samples containing representatives of various higher vertebrate taxa are to be analysed, the risk of false negatives strongly increases with decreasing universality of primers. As a consequence
we recommend the use of 16S as additional standard DNA barcoding marker for vertebrates, especially for but not limited to applications that involve pooled samples.
Acknowledgements
For comments, technical help and/or discussions we are grateful to Paul D. N. Hebert, Axel Meyer, Dirk Steinke, Diethard Tautz and David B. Wake. We are further indebted to Simone Hoegg, Pablo Orozco and Mario Vargas who provided help in the lab, and to the Madagascan authorities for research permits. The DNA barcoding project of Madagascan tadpoles was supported by a grant of the Volkswagen foundation to M Vences and to F Glaw. DR Vieites was supported by the AmphibiaTree project (NSF grant EF –O334939).
A previously unrecognised radiation of ranid frogs in Southern Africa revealed by nuclear and mitochondrial DNA sequences
Pyxicephalus adspersus
3. A previously unrecognised radiation of ranid frogs in Southern Africa revealed by nuclear and mitochondrial DNA sequences
Published:
Van der Meijden, A., M. Vences, S. Hoegg and A. Meyer. 2005. A previously unrecognized radiation of ranid frogs in Southern Africa revealed by nuclear and mitochondrial DNA sequences. Molecular Phylogenetics and Evolution 37: 674–685.
3.1 Abstract
In sub-Saharan Africa amphibians are represented by a large number of endemic frog genera and species of incompletely clarified phylogenetic relationships. This applies especially to African frogs of the family Ranidae. We provide a molecular phylogenetic hypothesis for ranids, including 11 of the 12 African endemic genera. Analysis of nuclear (rag-1, rag-2 and rhodopsin genes) and mitochondrial markers (12S and 16S ribosomal RNA genes) provide evidence for an endemic clade of African genera of high morphological and ecological diversity thus far assigned to up to five different subfamilies: Afrana, Cacosternum, Natalobatrachus, Pyxicephalus, Strongylopus, and Tomopterna. This clade has its highest species diversity in southern Africa, suggesting a possible biogeographic connection with the Cape Floral Region. Bayesian estimates of divergence times place the initial diversification of the southern African ranid clade at ~ 62-85 million years ago, concurrent with the onset of the radiation of afrotherian mammals. These and other African ranids (Conraua, Petropedetes, Phrynobatrachus, Ptychadena) are placed basally within the Ranoidae with respect to the Eurasian groups, which suggests an African origin for this whole epifamily.
Abbreviations: MYA, Million Years Ago; MP, Maximum Parsimony; ML, Maximum Likelihood; BI, Bayesian Inference; NJ, Neighbour Joining
3.2 Introduction
The recent report of the Global Amphibian Assessment project (Stuart et al., 2004) shows that at least a disturbing 42 percent of amphibian species are experiencing declines, in large part due to still unknown processes. In some cases entire diverse clades of frogs are heavily declining (Lötters et al., 2004). Such non-random extinctions can lead to a severe loss of evolutionary history (Purvis et al., 2000) and a reliable phylogeny of all amphibians is needed to identify them. In several very species-rich cosmopolitan groups of frogs the phylogenetic relationships are still insufficiently known. This lack of a robust phylogenetic hypothesis is especially true for the family Ranidae or True Frogs that contains over 700 species, which are distributed throughout the world. A single genus (Rana) is thought to occur on all continents except Antarctica. Yet the phylogenetic relationships among Rana, and ranids in general, are largely uncharted (Emerson et al., 2000). Recent molecular studies have provided important progress in the understanding of ranids and its related groups (Bossuyt and Milinkovitch, 2000; Hoegg et al., 2004a;
Van der Meijden et al., 2004; Vences et al., 2003b). Some studies have identified India as a reservoir of ancient ranid lineages, and proposed these animals as a model for "Out of India" dispersal of vertebrates (Bossuyt and Milinkovitch, 2001; Roelants et al., 2004).
These works demonstrated the potential of ranids to decipher general patterns of biogeography and diversification although only a part of the currently recognized ranid diversity has been studied so far. Because most of the endemic African ranid genera are still unstudied from a molecular perspective the biogeographical insights remain incomplete.
Despite recent compelling evidence for the ability for transoceanic dispersal in amphibians (Hedges et al., 1992; Vences et al., 2004; Vences et al., 2003b), there is little doubt that continental drift has had a major influence in shaping their current distribution and phylogeny. The close relationships of the recently discovered Nasikabatrachus from India with Nesomantis from the Seychelles strikingly demonstrated the importance of the Gondwanan breakup for the vicariance biogeography and hence phylogeny of these basal
