Molecular Phylogeny of Malagasy Reed Frogs, Heterixalus, and the Relative Performance of Bioacoustics and Color-Patterns for Resolving Their Systematics
Total Page：16
File Type：pdf, Size：1020Kb
Molecular phylogeny of Malagasy reed frogs, Heterixalus, and the relative performance of bioacoustics and color-patterns for resolving their systematics Katharina C. Wollenberg a,*, Frank Glaw b, Axel Meyer c, Miguel Vences a a Zoological Institute, Division of Evolutionary Biology, Technical University of Braunschweig, Spielmannstraße 8, 38106 Braunschweig, Germany b Zoologische Staatssammlung, Mu¨nchhausenstraße 21, 81247 Mu¨nchen, Germany c Lehrstuhl fu¨r Zoologie/Evolutionsbiologie, Department of Biology, University of Konstanz, 78457 Konstanz, Germany Abstract The members of the genus Heterixalus constitute one of the endemic frog radiations in Madagascar. Here we present a complete spe cies level phylogeny based on DNA sequences (4876 base pairs) of three nuclear and four mitochondrial markers to clarify the phylo genetic relationships among and within all known species of this genus, as well as the phylogenetic position of the monospecific Seychellean Tachycnemis seychellensis. Although the performance to resolve supported clades of Heterixalus species differed among the investigated gene fragments when analyzed separately, we could identify five well supported species groups within Heterixalus in the combined analysis of all gene fragments. Our data strongly support a Heterixalus Tachycnemis clade, and indicate the probable monophyly of Heterixalus placed sister to Tachycnemis. However, the diversification of these lineages may have happened in a short interval of time, leading to an unstable placement of Tachycnemis in the single gene fragment phylogenies. Referring to the hitherto exist ing classification of Heterixalus, which is predominantly based on chromatic and bioacoustic characters, we examined the relative per formance of these data sets relative to our molecular phylogeny. A Bayesian tree reconstructed with a bioacoustic data set yielded a higher resemblance to the molecular phylogeny than a tree constructed using a chromatic data set, which supports the importance of bioacoustic characters for systematic analyses of these anurans. Keywords: Amphibia: Anura: Hyperoliidae: Heterixalus; Madagascar; Molecular phylogeny; Systematics; Color patterns; Bioacoustics 1. Introduction molecular phylogenetic data support close relationships between Tachycnemis and Heterixalus, and the Heterixa- The treefrog family Hyperoliidae is composed of 18 lus Tachycnemis clade being deeply nested within African genera (Frost et al., 2006; AmphibiaWeb, 2006), all but hyperoliid genera, possibly sister to Afrixalus (Richards two of which are endemic to Africa. The two exceptions and Moore, 1996; Vences et al., 2003b; Frost et al., are Heterixalus, endemic to Madagascar and containing 2006). Molecular clock analyses indicated a relatively 11 species, and the monotypic genus Tachycnemis, with young divergence between African and Malagasy Sey- the only species T. seychellensis being endemic to the chellean hyperoliids at about 30 million years ago, much Seychelles islands (Drewes, 1984). Although morphologi- more recent than the Gondwanan connection between cal phylogenies placed Tachycnemis as a basal represen- these land masses that was severed about 160 130 mya tative of the family (Drewes, 1984; Channing, 1989), (Briggs, 2003). This indicates that colonization of Mada- gascar and of India by these frogs must have occurred * Corresponding author. Fax: +49 531 391 8198. by overseas dispersal during the Cenozoic (Vences E mail address: kc.wollenberg@tu bs.de (K.C. Wollenberg). et al., 2003c). 15 Despite the obvious biogeographic interest in under- known from only 32 out of 225 polychromatic frogs (Hoff- standing the relationships of the Malagasy Seychellean man and Blouin, 2000). Many of these are hyperoliids of hyperoliid radiation, a well-resolved phylogeny using mul- the genera Hyperolius and Heterixalus, and the hypothesis tiple markers and a dense taxon sampling is so far missing can therefore not be ruled out that their coloration might for this group. Their systematics was first established based be influenced by sexual selection. In species-level systemat- on morphology and color (Blommers-Schlo¨sser, 1982; ics, characters under sexual selection can be very useful in Blommers-Schlo¨sser and Blanc, 1991), and later refined phylogenetically assigning specimens to biological species. with bioacoustic analyses (Glaw and Vences, 1993). Based In contrast, when the phylogenetic relationships among on these data, three species groups are recognized (Glaw distantly related species are to be reconstructed, characters and Vences, 1993): (i) the H. betsileo group with H. betsi- under strong selection (be it sexual or natural) are unsuit- leo, (ii) the H. tricolor group with H. tricolor, H. variabilis able because they are likely to show high levels of homo- and H. andrakata, and (iii) the H. madagascariensis group, plasy. It furthermore has been suggested that in containing H. madagascariensis and H. boettgeri. At the aposematic animals color-patterns tend to be in greater time of the study of Glaw and Vences (1993), the calls of conflict with morphological signals concerning their phylo- H. rutenbergi and H. alboguttatus were not known yet, genetic significance than in non-aposematic animals (Are- H. carbonei and H. punctatus were only subsequently ekul and Quicke, 2006). Although some Heterixalus have described, and H. luteostriatus was not yet considered a a rather vivid coloration, it is very unlikely that this consti- valid species (Vences et al., 2000). Here we present a molec- tutes an aposematic signal. Conclusive statements on the ular phylogeny of all known Heterixalus species and of vivid and often dimorphic color of these frogs require fur- Tachycnemis based on an analysis of four mitochondrial ther experimental testing. and three nuclear gene fragments. The two major goals in In contrast, there is no doubt that the advertisement this study were: (1) to achieve a better understanding of calls of frogs are the main mechanism of mate recognition the relationships and of the genetic differentiation within and sexual selection in this amphibian group. Bioacoustic Heterixalus as a contribution to a future taxonomic revi- characters are of excellent use in taxonomy, although the sion of these frogs, and (2) to obtain molecular evidence rate in which different call parameters evolve can vary for the monophyly or non-monophyly of Heterixalus with among groups (Ryan, 1986; Cocroft and Ryan, 1995; Rich- respect to Tachycnemis. ards, 2006) and their value for phylogenetic reconstruction Anurans are known to have a high level of morpholog- is poorly understood. ical similarity and, often, homoplasy (e.g., Emerson, 1986) In reference to the hitherto existing classification of that has substantially obscured their systematics before the Heterixalus, being predominantly based on chromatic advent of molecular characters (e.g., Bossuyt and Milin- and bioacoustic characters, a third goal of this study was kovitch, 2000; Van der Meijden et al., 2005; Frost et al., to examine the performance of these characters to resolve 2006). Compared to most other anurans, hyperoliids of the phylogenetic relationships among populations and spe- several genera show even lower levels of morphological cies of Heterixalus as they were recovered using the molec- divergence, and taxonomy in the past has therefore largely ular data sets. We use the molecular phylogeny as a basis of been based on color-patterns. In Heterixalus, Glaw and comparison to evaluate the systematic relevance of bioa- Vences (1993) considered morphometrics as not very infor- coustic and chromatic characters for the identification of mative for distinguishing Heterixalus species, and restricted species and major clades within Heterixalus. taxonomically informative characters to body size, adver- tisement calls, and color. However, color-pattern is known 2. Materials and methods to be highly polymorphic within species of other hyperoliid genera, e.g., Hyperolius (Schiøtz, 1999; Amiet, 2004). Fur- We obtained DNA-sequences of 34 individuals from all thermore, the color-patterns in some species undergo per- currently valid 11 Heterixalus species and one Tachycnemis manent ontogenetic color changes (Richards, 1982). They seychellensis (Electronic Supplement Table E1), with multi- are known to be under hormonal control (e.g., Hyperolius ple populations from much of the known range sampled argus, Hayes and Menendez, 1999), which is related to sex- for most species. We analyzed one fragment of the mito- ual dimorphism. Intraspecific and intersexual color-pattern chondrial 12S rRNA, Cytochrome b (Cytb) and Cyto- polymorphism also occurs in Heterixalus(H. alboguttatus chrome Oxidase Subunit 1 genes (COI), two fragments of and H. variabilis, Glaw and Vences, 1994; Schmidt et al., the 16S rRNA gene, and one fragment each of the nuclear 2001). Tyrosinase, Rhodopsin and Rag-1 genes. In total, this Color and/or pattern are known to be important for yielded 4876 basepairs of sequence data (Table E2). The mate recognition or sexual selection in many taxa such as full alignment was submitted to TreeBase (http:// birds or in cichlid fishes (Salzburger and Meyer, 2004). www.treebase.org; Sequences were submitted to GenBank Although in frogs there is little evidence for female choice (accession numbers EF646474 EF646728; Table E1, Ben- based on male color-patterns, some indications for visual son et al., 2004). For primer information, see Table E2. color-based mate recognition may exist in diurnal poison PCR was performed