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Research article Open Access The importance of comparative phylogeography in diagnosing introduced species: a lesson from the seal salamander, Desmognathus monticola Ronald M Bonett*1, Kenneth H Kozak2, David R Vieites1, Alison Bare3, Jessica A Wooten4 and Stanley E Trauth3

Address: 1Museum of Vertebrate Zoology and Department of Integrative Biology, University of California at Berkeley, Berkeley, CA, 94720, USA, 2Department of Ecology and Evolution, Stony Brook University, Stony Brook, NY, 11794, USA, 3Department of Biological Sciences, Arkansas State University, State University, AR, 72467, USA and 4Department of Biological Sciences, University of Alabama, Tuscaloosa, AL, 35487, USA Email: Ronald M Bonett* - [email protected]; Kenneth H Kozak - [email protected]; David R Vieites - [email protected]; Alison Bare - [email protected]; Jessica A Wooten - [email protected]; Stanley E Trauth - [email protected] * Corresponding author

Published: 7 September 2007 Received: 25 February 2007 Accepted: 7 September 2007 BMC Ecology 2007, 7:7 doi:10.1186/1472-6785-7-7 This article is available from: http://www.biomedcentral.com/1472-6785/7/7 © 2007 Bonett et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Background: In most regions of the world human influences on the distribution of flora and fauna predate complete biotic surveys. In some cases this challenges our ability to discriminate native from introduced species. This distinction is particularly critical for isolated populations, because relicts of native species may need to be conserved, whereas introduced species may require immediate eradication. Recently an isolated population of seal salamanders, Desmognathus monticola, was discovered on the Ozark Plateau, ~700 km west of its broad continuous distribution in the Appalachian Mountains of eastern North America. Using Nested Clade Analysis (NCA) we test whether the Ozark isolate results from population fragmentation (a natural relict) or long distance dispersal (a human-mediated introduction). Results: Despite its broad distribution in the Appalachian Mountains, the primary haplotype diversity of D. monticola is restricted to less than 2.5% of the distribution in the extreme southern Appalachians, where genetic diversity is high for other co-distributed species. By intensively sampling this genetically diverse region we located haplotypes identical to the Ozark isolate. Nested Clade Analysis supports the hypothesis that the Ozark population was introduced, but it was necessary to include haplotypes that are less than or equal to 0.733% divergent from the Ozark population in order to arrive at this conclusion. These critical haplotypes only occur in < 1.2% of the native distribution and NCA excluding them suggest that the Ozark population is a natural relict. Conclusion: Our analyses suggest that the isolated population of D. monticola from the Ozarks is not native to the region and may need to be extirpated rather than conserved, particularly because of its potential negative impacts on endemic Ozark stream salamander communities. Diagnosing a species as introduced may require locating nearly identical haplotypes in the known native distribution, which may be a major undertaking. Our study demonstrates the importance of considering comparative phylogeographic information for locating critical haplotypes when distinguishing native from introduced species.

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Background to the east (Figure 1B). This isolated population repre- Species introduced by human activities are one of the sents either the only known, and possibly last, remnant of leading threats to biodiversity [1-4]. A critical step in an entire lineage (Desmognathus) in the Ozark Plateau or ameliorating the impacts and spread of introduced species an introduced species that could threaten local endemic is to identify and contain them in their infancy [5,6]. species if it spreads (see Discussion). However, humans have been altering biotic patterns across the world since the "Age of Exploration" [7], and There is a wealth of biogeographic evidence that suggests for many regions species introductions precede complete a relatively recent faunal connection between the Ozarks biodiversity inventories, obscuring our ability to distin- and the Appalachians, and many conspecifics or sister guish introduced from native flora and fauna. This is species of fish [30-32], crayfish [33] and salamanders potentially a very important yet time sensitive distinction, [11,34,35] occur across these regions. On the other hand, especially for isolated populations, because the alternate surveys have shown D. monticola to be one of the most diagnoses suggest opposite conservation action. Isolated common salamander species found in fishing bait shops native populations may need special protection [8-10], [36], so this population may have been introduced by while introduced species may need to be eliminated [5,6]. fishermen or by other human activities. Without further In the absence of historical information genetic data may detailed population genetic analyses, it is not possible to be necessary to determine the origin of an isolated popu- support or reject either scenario. lation, although pin-pointing the closest relatives in the known native distribution (to test the history of the iso- Using NCA, based on a portion of the mitochondrial gene late adequately) can be an endless task. Here, we show cytochrome oxidase-1 (cox1), we test whether this isolate that using comparative phylogeographic patterns of taxa is the result of a recent human-mediated introduction or that are co-distributed across the known, native range of a represents a relict from historical faunal connections putatively introduced population may be the most effec- between the Appalachian Mountains and the Ozark Pla- tive strategy for distinguishing native from introduced teau. If the Ozark population originated through fragmen- species. tation and contraction of a previously more widespread distribution, then we would expect it to exhibit significant The Plethodontidae is the most species rich family of sal- genetic divergence from Appalachian populations. Alter- amanders [11,12], and their diversity has been primarily natively, if the Ozark population results from a recent shaped by allopatric speciation events resulting in an introduction through human activities, we would expect abundance of cryptic species and genetically distinct iso- little or no genetic divergence from Appalachian popula- lated populations [e.g., [13-16]. The plethodontid genus tions, despite the large geographic separation between Desmognathus, endemic to eastern North America, these highland regions. These predictions are ideally includes 19 recognized species; their primary diversity is suited for testing with Nested Clade Analysis, which uses centered in the southern portion of the Appalachian the geographic distributions of ancestral (interior) haplo- Mountains [11,12] (Figure 1A). This genus is marked by types relative to younger (tip) ones to draw inferences great ecomorphological diversity, ranging from small, about processes that have shaped spatial patterns of strictly terrestrial species to very large-bodied, stream- genetic variation. dwelling predators [17-19], but most ecomorphs also contain parapatrically distributed cryptic species [20-22]. Adequately sampling genetic diversity is critical for testing phylogeographic hypotheses [37,38]. Although, given the The Ozark Plateau of east-central North America is a karst fact that genetic diversity is not always randomly distrib- uplift separated from the Appalachian Mountains by the uted across a species' distribution, it can be challenging to low elevation flood plain of the Mississippi River. The assess sampling adequacy a priori. Our sampling includes Ozark Plateau harbors many endemic species, including a the entire latitudinal distribution of D. monticola, with unique salamander fauna [23,24]. There are only a few dense sampling from the southernmost extent of the historical reports of Desmognathus on the Ozark Plateau Appalachian Mountains where lineage diversity is known [25,26], but no credence has been given to these records to be high for other co-distributed taxa [39-43]. We find [27,28] since no museum vouchers exist and no subse- that the highest genetic diversity in D. monticola is quent specimens were found for the next 40+ years. In restricted to a localized region (< 2.5% of the distribution) 2003, a very restricted, but thriving, population of in northern Georgia, and a correct diagnosis of the Ozark Desmognathus was discovered in the western Ozarks [29]. population is highly dependent on including critical hap- This population is similar, both morphologically and lotypes from this region in the NCA. We discuss our mitochondrially, to the seal salamander (Desmognathus results in the context of developing sampling strategies monticola), a large stream-dwelling species that is wide- based on comparative phylogeography of co-occurring spread in the Appalachian Mountains, more than 700 km

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FigureDistribution 1 and species richness of Desmognathus FigureGeographicnetworks 2 for distribution D. monticola and haplotypes unrooted statistical parsimony Distribution and species richness of Desmognathus. A. Geographic distribution and unrooted statistical par- Species richness of the genus Desmognathus, with the highest simony networks for D. monticola haplotypes. The species diversity in the southern Appalachian Mountains. large majority of the geographic distribution contains only a Note: Desmognathus are absent from the Ozark Plateau. B. few haplotypes (K, L, M, N, O, & P). The Ozark haplotype (F, Geographic distribution of Desmognathus monticola outlined red) occurs in the southern Appalachians of northern Geor- in blue overlaid on an elevation map of eastern North Amer- gia amongst a great diversity of related haplotypes. Labels on ica. The isolated population of D. monticola in the Ozarks is the network indicate the haplotype and the number of coun- designated with a blue star. Inset is a photograph of an adult ties where it was found (sizes of circles are also drawn pro- D. monticola from the Ozarks. portional to this number). Colors of haplotypes correspond to pie diagrams on the map and show the frequency of haplo- tyope in each county sampled. Black dots on haplotype net- work indicate hypothetical unsampled haplotypes. species when assessing the history of populations that are suspected of being introduced. Ozark population (haplotype F) were found in the region Results of highest haplotype diversity in northeast Georgia. The There was no nucleotide variation in the 515 basepair origin of the Ozark population of D. monticola was inves- (bp) cox1 fragment among our seven Ozark samples. The tigated with NCA [44,45] (See Methods Section). The dis- 100 Appalachian samples from 47 populations covering tance among populations that have haplotype F is the entire latitudinal distribution of D. monticola repre- significantly large (Dc = 310.58, Dn = 292.71; Figure 3). sented 18 unique cox1 haplotypes (Additional File 1). The The distance between haplotype F and its nearest neigh- geographic distribution of haplotypes was highly dispro- bor in the network (haplotype C) is also significantly large portional, with the highest diversity centered in the south- (Dc = 251.27, Dn = 217.20). NCA of the clade including ern Appalachian Mountains (Figure 2, Table 1). haplotypes C and F (clade 2-2) indicates that the Ozark Appalachian haplotypes ranged from 0.000% to 2.689% population is the result of long distance colonization (i.e., divergent from the Ozark population. Haplotypes identi- Introduction). Our path to this conclusion is as follows. cal (zero mutational steps; 0.000% divergent) to the All nested clades are from separate areas (i.e., there is no

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Table 1: Comparison of cox1 divergence between Appalachian only identical Appalachian haplotypes (Haplotype F = and Ozark haplotypes. The Ozark population only contains a 0.56% of the distribution), still leads to the inference that single haplotype (F). Note the large number of haplotypes restricted to northern Georgia (GA). See Figure two for map and the Ozark population was introduced. However, exclud- Additional File 1 for locality details. ing Appalachian haplotypes F, C, and I, leads to a signifi- cantly small Dc for clade 2-2 (i.e., Ozark haplotype F). Haplo uncorrected p to the Number States Since the Ozark population is separated from the remain- type Ozark population of localities ing haplotypes by a larger than average number of muta- tional steps, and is allopatric from clades 2–3 and 2–4, A1.222%1GA this leads to the inference of allopatric fragmentation at B1.711%1GA the level of clade 3–2. This demonstrates that in this case C0.733%2GA it is necessary to locate and include in the NCA, haplo- D1.956%1GA types that are ≤ 0.733% divergent from the Ozark popula- E1.467%4GA tion, in order to properly diagnose the Ozark population F 0.000% 5 AR, GA G0.978%1GA as introduced. The distributions of these critical haplo- H0.978%1GA types (F, C, and I) are localized to a small region in north- I0.733%1GA ern Georgia that comprises < 1.2% of the geographic J0.978%1GA distribution of D. monticola (Figure 4). K1.956%1GA L1.467%1VA Discussion M1.711%2TN Distinguishing native populations from human N 1.467% 1 NC O1.467%1KY introductions P 1.222% 13 GA, KY, NC, PA, To date most genetic studies of introduced species have TN, VA, WV focused on evolutionary genetics [48,49], genetic diversity Q 2.934% 2 AL, GA [50], hybridization [51,52], and the identification of R2.689%1GA source populations of species that are known to be intro- duced [e.g., [50,53-57]. The field of phylogeography has State abbreviations are as follows: played a major role in understanding patterns of species AL = Alabama, AR = Arkansas, GA = Georgia, KY = Kentucky, NC = North Carolina, PA = Pennsylvania, TN = Tennessee, VA = Virginia, invasions [51,58]. However, there are relatively few stud- and WV = West Virginia. ies that have tested whether or not a given species has been introduced by human activities [59-62], and little geographic overlap between the distribution of haplo- attention has been given to the approaches and pitfalls of types F and C; Yes to couplet #1). The species is present molecular-based identification of putative invaders between where haplotypes F and C were found within the [63,64]. Here we demonstrate a case where the correct Appalachians (Yes to couplet #19), and we did sample diagnosis of an isolated population (as native or intro- this area (Yes to couplet #20). None of the conditions in duced) using molecular methods is highly dependent on couplets #2 or #11 are satisfied. There is a reversal in sig- locating and sufficiently sampling a very restricted portion nificance between I-T Dn and Dc values (I-T Dn = -178 S, of the native distribution of a wide-ranging species. We Dc = 251.27 L; Yes to couplet #12). Since the Ozark pop- identified this region and sampled it intensively based on ulation is separated from the geographical center of other the biogeographic history and patterns of genetic diversity clades (Yes to couplet #13), and there are no mutational of other co-distributed fauna. differences between this population and some Appala- chian populations, we conclude that the Ozark popula- Phylogeographic analyses based on inadequate sampling tion arose from long distance colonization. Given the can lead to spurious results [37,38]. For many reasons, NCA evidence for restricted gene flow and dispersal patterns of genetic variation are often non-randomly dis- within portions of the native range, and the limited dis- tributed across the species landscape. Therefore, the per- persal capabilities of plethodontid salamanders [46,47], centage of geographic distribution sampled does not this long-distance movement likely resulted from human necessarily equate to the percentage of existing haplotypes activities. sampled, making it difficult to determine when adequate sampling has been achieved. Examining patterns of In order to test the necessity of including very similar (or genetic diversity of co-distributed species can provide identical) haplotypes to determine whether the Ozark clues to patterns of diversity in the species of interest [65]. population is native or introduced, we sequentially ran Many eastern North American species show considerable additional NCAs that excluded Appalachian samples that concordance in phylogeographic patterns, particularly were 0.000% (haplotype F), and ≤ 0.733% (haplotypes F, due to the effects of Pleistocene glaciation [reviewed in C, and I) divergent from the Ozark population. Excluding [39]]. One phylogeographic pattern common to many

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GraphicalFigure 3 summary of the nested haplotype structure and NCA Graphical summary of the nested haplotype structure and NCA. Individual haplotypes are listed across the top, with increasingly more inclusive nested groups extending to the bottom. Interior haplotypes/nested groups are depicted in bold ital- ics. Significant DC, DN, and I-T values are reported. Distances that are significantly small or large are indicated with S or L, respectively. The path taken through the most recent version of the inference key is shown; RGF, restricted gene flow; PF, past fragmentation; LDC, long-distance colonization; RE w/PF, range expansion coupled with past fragmentation. NCA results directly relevant to the diagnosis of the Ozark population (haplotype F) are highlighted in blue. animals, particularly salamanders, is that southern popu- nant that deserves conservation attention (Figure 4). An lations represent historical refugia and are genetically analysis that contains a broad sampling from across the diverse while more northern populations underwent known native distribution, but only missing these critical recent post-glacial range expansions and are often geneti- haplotypes could look convincing, and subsequently lead cally uniform [e.gs., [20,39-43]. The phylogeographic pat- to an incorrect diagnosis and the protection of an intro- terns that we have elucidated for D. monticola are duced species. consistent with patterns exhibited by many co-distributed species. The large majority of the distribution of D. monti- Comprehensive sampling of genetic diversity of the spe- cola represents a single lineage (containing the closely cies' known native distribution may be essential for mak- related haplotypes K, L, M, N, O, and P) that likely repre- ing an accurate assessment of the history of populations as sents a recent range expansion from the southernmost native or introduced by humans, but exhaustive sampling highlands of the Appalachian Mountains all the way to may not be practical, especially when rapid identification the most northern extent of the distribution (Figure 2). is necessary for conservation strategies to be immediately The southern portion of the distribution may be a histor- implemented. We suggest that considering comparative ical refuge and thus contains the highest genetic diversity. phylogeographic patterns of co-distributed species can More than 50% of the cox1 haplotype diversity of D. mon- expedite an accurate diagnosis by acting as a guide to sam- ticola is restricted to less than 2.5% of the current geo- pling the known native distribution of the species in ques- graphic distribution of the species. In this case, the source tion. For species that have undergone recent range of the human-mediated Ozark introduction is derived expansions such as the one in this study, a comparative from this genetically diverse region in the southern Appa- phylogeographic approach can help locate the areas of lachian Mountains. When analyzing the isolated Ozark highest genetic diversity, and prevent over-sampling in population of D. monticola in terms of a broad geographic genetically uniform regions. Furthermore, if phylogeo- sampling of its known native distribution, but only graphic data are available for co-distributed species with excluding Appalachian haplotypes that are ≤ 0.733%, the ecological requirements that are similar to those of the Ozark population appeared to be a unique historical rem- species in question (i.e., taxa potentially influenced by

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PercentFigure 4divergence of Appalachian haplotypes to the Ozark population and their geographic distribution Percent divergence of Appalachian haplotypes to the Ozark population and their geographic distribution. Inset graph depicts the % sequence of the Appalachian haplotypes to the Ozark population and the % of the area of the distribution that they occur. The map shows the genetic landscape of haplotypes in relation to their divergence from the Ozark population. Note that it is necessary to include haplotypes that are ≤ 0.733% divergent from the Ozark population in order to diagnose it as introduced. similar patterns of vicariance), this could effectively guide cox1 haplotypes with populations of D. monticola from further sampling. For example, if the known native distri- four counties in the northeastern corner of the state of bution of a terrestrial species extends across a region that Georgia in the southern Appalachians (~1000 km away). is dissected by many rivers that are barriers to gene flow Nested Clade Analysis of the cox1 fragment shows that this for other co-distributed terrestrial species, then initial highly disjunct population results from a long distance analyses could compare the population in question to dispersal (human-mediated introduction). Furthermore, individuals from the land between each river (i.e., to each we sequenced an additional 1.6 kb of a more variable primary biogeographic subunit). This could help to nar- region of mitochondrial DNA, upstream from our cox1 row down which part of the distribution is genetically fragment, for select individuals. This region includes the most similar to populations in question, and could help gene NADH dehydrogenase subunit 2, tRNATrp, tRNAAla, to focus subsequent sampling. tRNAAsn, tRNACys, tRNATyr, the origin for light-strand rep- lication, and the beginning of cox1. We found only a sin- Introduced Ozark Desmognathus gle nucleotide substitution between the Ozark population Based on our analyses it appears that the population of D. and our samples from northeast Georgia (i.e., in total only monticola recently discovered in the Ozark Plateau is intro- one substitution in > 2 kb of mitochondrial DNA). duced. We found that this population shares identical

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How did a southern Appalachian salamander get trans- adults would likely be the competitively-superior sala- ported to the western Ozarks? In the southeastern United manders in a spring or stream. Although predation by States, stream-dwelling plethodontid salamanders of the large stream-dwelling Desmognathus on other salamanders genera Desmognathus, Gyrinophilus, and Pseudotriton are in the wild is rare [79], this species has been shown to dis- vernacularly referred to as "spring lizards", and are com- place heterospecifics from the most suitable moist habi- monly used as fishing bait for catching large species of tats [80]. Therefore, the presence of D. monticola could centrarchid fishes such as largemouth bass (Micropterus have a negative impact on Ozark Eurycea simply by salmoides). "Spring lizards" are collected from the wild excluding them from moist habitats at the periphery of and sold in bait shops by the thousands [66]. In fact, sur- springs and streams, a habitat feature that can be quite veys of bait shops in northern Georgia (the source area of limited in arid areas of the Ozark Plateau. the Ozark introduction) found D. monticola to be the most common salamander sold [36]. Unfortunately, there is lit- Ozark Desmognathus monticola are currently known from tle information on the broader sales and distribution of only two locations. The salamanders included in this "spring lizards" to investigate whether a bait bucket was study are from a small spring ca. 1 meter wide that issues the source of the Ozark introduction. This is not the only from the underside of a dirt bank and flows approxi- long-distance introduction of a salamander in the United mately 8.5 meters before reaching a much larger stream States. North American tiger salamanders such as the (Spavinaw Creek). Since the original discovery in 2003 we barred tiger salamanders (Ambystoma mavortium) are also have visited this site four times and have found up to 25 extensively collected and sold as fishing bait in the central individuals, ranging in size from very small newly meta- and western United States. Populations of A. mavortium morphosed juveniles, to very large adults (75+ mm SVL), have been introduced to localities in California (~2000 in a single survey. Among the largest individuals, we km from their native distribution) where they hybridize found one gravid female, 55 mm SVL, with enlarged (ca. with the rare California tiger salamander, Ambystoma cali- 2.5–3.0 mm in diameter) ovarian follicles (ca. 35 ova). forniense [52]. Another case is of the wandering salaman- Therefore, it appears that D. monticola are breeding and der, Aneides vagrans, which was accidentally introduced to reproducing at this location. Recently, a second Ozark D. western Canada from central California through ship- monticola location was discovered by members of the ments of bark from oak trees in the early 1900s [60]. Arkansas Herpetological Society. Three adult D. monticola These salamanders have become well established and very were found in a small spring entering Spavinaw Creek widespread in coastal British Colombia and Vancouver, (36.4030°N, 94.3569°W) approximately 2.5 km but are not noted to be invasive [60]. upstream of the locality included in our analyses (K. Rob- erts personal communication 2006). We presently know lit- Do introduced Desmognathus monticola have a negative tle about the abundance of Desmognathus monticola in the ecological impact in the Ozark Plateau? The Ozark Plateau Ozarks and additional research on the ecological interac- is home to four stream/spring-dwelling species of pletho- tions between this introduced species and co-occurring dontid salamanders of the genus Eurycea:E. lucifuga, E. species of Eurycea are necessary to determine the level of longicauda melanopleura, E. spelaea, and E. tynerensis, and impact that it has on local salamander fauna. the latter two (E. spelaea and E. tynerensis) are actually spe- cies groups that represent a major endemic Ozark radia- Conclusion tion [24]. Although stream-dwelling Desmognathus and Our analyses of mitochondrial DNA indicate that the Eurycea are broadly sympatric in the Appalachian and highly disjunct population of seal salamanders, Desmog- Ouachita Mountains, the Ozark Plateau is geologically nathus monticola, recently discovered on the Ozark Pla- distinctly different from these regions [67] and contains teau, is not native to the region. This population may need very different stream habitats [68-70]. The Ozark Plateau to be extirpated because of its potential negative impacts is well drained and is very dry during the summer months. on endemic Ozark stream-dwelling salamanders. This Moist streamside habitats that are important for some conclusion could only be realized once nearly identical metamorphosing species of Eurycea as well as D. monticola haplotypes from the known native distribution were are quite limited. Desmognathus monticola are large territo- included in our analyses. Locating the most genetically rial salamanders and are very aggressive towards both similar individuals (the putative source population) in conspecific and heterospecific stream-dwelling salaman- the known native distribution of a species to effectively ders [71]. It has been hypothesized that in the southern test the origin of an isolated population can be a labor- Appalachians, where up to seven species of Desmognathus intensive and costly undertaking. We propose that consid- occur sympatrically [72], ecological boundaries between ering comparative phylogeographic patterns can greatly congeners are maintained by aggressive interference and facilitate this process and expedite diagnoses of native and predation [73-78]. Desmognathus monticola is larger and introduced species. more robust than any of the Ozark Eurycea, and large

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Methods Nested clade analysis Sampling The origin of the Ozark population of D. monticola was Salamanders from the Ozark population were collected investigated with Nested Clade Analysis [44,45] in order from 30-September-2003 to 4-April-2005. Due to the lim- to test between two alternate hypotheses: past fragmenta- ited distribution of the Ozark Desmognathus, vouchers tion (a natural historical relict) or long distance coloniza- were only taken for the first six individuals and were tion (human-mediated introduction). Haplotype deposited at Arkansas State University Museum of Verte- networks were constructed with statistical parsimony brates (ASUMZ 28083–28086; 28156; 29032). Tail tips method implemented in TCS [86], and the haplotype net- were taken in the field from subsequent specimens works were converted into nested clades [87,88]. The because at the time we did not know the species was intro- coordinates used in the analyses were either determined duced. These were compared to a large database of D. with a Garmin XL Global Positioning System (GPS) in the monticola cox1 sequences from GenBank [81,82], speci- field, or extrapolated from topographical maps. The only mens collected from 8-May-2000 to 16-May-2002 by KHK locality information available for some of the GenBank that were deposited in the University of Minnesota's John sequences was the state and county, so we estimated the Ford Bell Museum (JFBM), two tissue samples from the coordinates of the center point of the county for those Museum of Vertebrate Zoology, University of California, samples. GeoDis version 2.0 [89] was used to calculate (1) Berkeley (MVZ), and samples collected in northern Geor- the clade distance Dc, which measures the average dis- gia by JAW from 12-January-2006 and 26-March-2006 tance of haplotypes in a nested group from its geographi- that were deposited in the MVZ (Additional File 1). cal center, (2) the nested clade distance Dn, which measures how far a haplotype group is from the geo- DNA isolation, amplification and sequencing graphic center of other groups with which it is nested, and DNA was extracted from fresh frozen tissues and ethanol 3) the average Dc and Dn separating interior and tip preserved tissues using Qiagen DNeasy extraction kits. A groupings of haplotypes. To test whether the geographic portion of the mitochondrial gene cytochrome oxidase 1 distributions of haplotypes were more widespread or (cox1, 515 bp not including primers) was amplified using restricted than expected by chance, we used a categorical the primers COX1F (5'-GGTATTGAGGTTTCGGTCTG-3') permutation contingency analysis. The most likely histor- and COX1R (5'-CTTAGTCTCTTAATTCGAGC-3') [81] ical and recurrent processes responsible for statistically with standard protocols. PCR products were run out on significant patterns of phylogeographic variation were 1.5 % agarose gels. Successful amplifications were cleaned inferred using the revised inference key 11 November with a Millipore PCR96 cleanup kit (Montáge™) or EXOS- 2005 [45]. APIT (USB Corp.). Big Dye (ABI) was used for cycle sequencing reactions sequenced on an ABI 3730 capillary Abbreviations sequencer. Sequencher ™ 3.1 (Gene Codes Corp.) was ASUMZ, Arkansas State University Museum of Zoology; used to align and edit sequences. The alignment was bp, basepairs; cox1, cytochrome oxidase 1; Dc, clade dis- unambiguous and the translation was checked in Mac- tance; Dn, nested clade distance; I-T, interior to tip; JFBM, Clade [83]. Sequences were deposited in GenBank; acces- John Ford Bell Museum; MVZ, Museum of Vertebrate sion numbers are provided in Additional File 1. For the Zoology; NCA, nested clade analysis; PF, past fragmenta- analyses we trimmed the fragment to 409 bp so that we tion; RE, range expansion; RGF, restricted gene flow. could utilize sequences from GenBank. PAUP* 4.0b10 [84] was used to calculate uncorrected pairwise sequence Authors' contributions divergences among populations. The sizes of the whole RMB coordinated the study, collected and analyzed distribution of D. monticola, was based on the Global sequence data, and primarily prepared the manuscript. Amphibian Assessment [85]. We omitted a disjunct iso- KHK contributed many Appalachian samples, performed late from southern Alabama because preliminary mito- the NCA, and contributed to the preparation of the man- chondrial DNA evidence suggests that it is a different uscript. DRV assisted in collecting sequence data and con- species [81,82]. The size and percentage of the distribu- tributed to the focus and preparation of the manuscript. tion of selected haplotypes was estimated based on the AB discovered the Ozark population of D. monticola and size of the county where they occur. This is actually a slight participated in subsequent field trips. JAW collected inval- over estimate as multiple haplotypes occur in some coun- uable material from across the state of Georgia and edited ties, so in this species locating specific haplotypes to prop- the manuscript. SET made the initial identification of AB's erly diagnose the introduction should be at least as discovery and coordinated additional fieldwork and con- difficult as we suggest if not more. tributed to the preparation of the manuscript. All authors read and approved the final manuscript.

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Additional material species formation. Proceedings of the National Academy of Sciences USA 2000, 97:1640-1647. 14. Chippindale PT: Species boundaries and species diversity in the central Texas hemidactyliine plethodontid salamanders, Additional file 1 genus Eurycea. In The Biology of Plethodontid Salamanders Edited by: Locality information, museum and Genbank accession numbers for the D. Bruce RC, Jaeger RG, Houck LD. (New York, USA): Kluwer Aca- monticola samples used in this study. demic, Plenum Publishers; 2000:149-165. Click here for file 15. Kozak KH, Weisrock DW, Larson A: Rapid lineage accumulation in a non-adaptive radiation: Phylogenetic analysis of diversi- [http://www.biomedcentral.com/content/supplementary/1472- fication rates in eastern North American woodland salaman- 6785-7-7-S1.doc] ders (Plethodontidae: Plethodon). Proceedings of the Royal Society of London B 2006, 273:539-546. 16. Wake DB: Problems with species: patterns and processes of species formation in salamanders. Annals of the Missouri Botanical Garden 2006, 93:8-23. Acknowledgements 17. Hairston NG: Species packing in the salamander genus We thank Kelly Irwin of the Arkansas Game and Fish for facilitating the Desmognathus : what are the interspecific interactions involved? The American Naturalist 1980, 115:354-366. investigation of the Ozark population and issuing collecting permits. We are 18. Bruce RC: Life-history perspective of adaptive radiation in grateful to D. Buckley, C. Camp, B. Cockrum, S. Eagle, I. Martínez-Solano, desmognathine salamanders. Copeia 1996, 1996:783-790. and B. Wheeler for joining us in the field during collecting. Additional tis- 19. Kozak KH, Larson A, Bonett RM, Harmon LJ: Phylogenetic analy- sues samples used in this study were provided by the Museum of Verte- sis of ecomorphological divergence, community structure, and diversification rates in dusky salamanders (Plethodonti- brate Zoology and we thank Brian Lin was for his help in the lab at the MVZ. dae: Desmognathus). Evolution 2005, 59:2000-2016. We thank Paul Chippindale for use of his lab at the University of Texas at 20. Tilley SG, Mahoney MJ: Patterns of genetic differentiation in sal- Arlington where RMB collected a couple preliminary sequences. We also amanders of the Desmognathus ochrophaeus complex thank K. Roberts and the Arkansas Herpetological Society for sharing infor- (Amphibia: Plethodontidae). Herpetological Monographs 1996, 10:1-42. mation on the additional Ozark D. monticola location that they recently dis- 21. Bonett RM: Analysis of the contact zone between Desmog- covered. S. Emel, E. Timpe, M. Tumeo, D. Wake, M. 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These are an electronic appendices to the paper by Vences et al. 2003 Multiple overseas dispersal in amphibians. Proc. R. Soc. Lond. B 270, 2435–2442. (doi:10.1098/rspb.2003.2516)

Electronic appendices are refereed with the text. However, no attempt has been made to impose a uniform editorial style on the electronic appendices.

Table of Contents

Appendix A. Primers used for DNA amplification and sequencing

Appendix B. Voucher specimens and Genbank accession numbers

Appendix C. Substitution models used in Maximum Likelihood Phylogenetic analysis

Appendix D. Details of Bayesian phylogenetic analysis

Appendix E. Separate analysis of gene fragments

Appendix F. Testing alternative topologies with Shimodaira-Hasegawa tests

Appendix G. Estimates of divergence times

Appendix H. A survey of the terrestrial and freshwater vertebrate families of Madagascar, their biogeographic relationships and fossil ages

Appendix I. References used in electronic appendices

Appendix A. Primer sequences used for DNA amplification and sequencing

All primers are given in 5'-3' direction. Primers marked with an asterisk were newly developed for this paper.

Cytochrome b

F-Primer 1 (1): CBJ10933 - TAT GTT CTA CCA TGA GGA CAA ATA TC

F-Primer 2 (1): Cytb-a - CCA TGA GGA CAA ATA TCA TTY TGR GG

*F-Primer 3: MVZ15L-mod - AAC TWA TGG CCC MCA CMA TMC GWA A

R-Primer 1 (1): Cytb-c - CTA CTG GTT GTC CTC CGA TTC ATG T

*R-Primer 2: CytbAR-H-mod - TAW ARG GRT CYT CKA CTG GTT G

Tyrosinase (exon 1)

F-Primer 1 (1): Tyr-1b - AGG TCC TCY TRA GGA AGG AAT G

F-Primer 2 (1): Tyr-1d - TCC TCC GTG GGC ACC CAR TTC CC

F-Primer 3 (1): Tyr-1a - AGG TCC TCT TRA GCA AGG AAT G

*F-Primer 4: Tyr-F40 - AAR GAR TGY TGY CCI GTI TGG

*F-Primer 5: Tyr - Fx3 - ACT GGC CCA YTG THT TYT ACA AC

*F-Primer 6: Tyr - Fx4 - YTG GCC YWY TGT NTT YTA YAA C

R-Primer 1 (1): Tyr-1g - TGC TGG CRT CTC TCC ART CCC A

R-Primer 2 (1): Tyr-1e - GAG AAG AAA GAW GCT GGG CTG AG *R-Primer 3: Tyr-SPA - GAI GAG AAR AAR GAI GCT GGG CT

Rhodopsin (exon 1)

*F-Primer 1: Rhod-ma - AAC GGA ACA GAA GGY CC

F-Primer 2 (1): Rhod-1a - ACC ATG AAC GGA ACA GAA GGY CC

*R-Primer 1: Rhod-md - GTA GCG AAG AAR CCT TC

R-Primer 2 (1): Rhod-1d - GTA GCG AAG AAR CCT TCA AMG TA

R-Primer 3: Rhod-1c - CCA AGG GTA GCG AAG AAR CCT TC

12S rRNA & tRNAVal

F-Primer 1 (2): 12SAL - AAA CTG GGA TTA GAT ACC CCA CTA T

R-Primer 1 (2): 12SBH - GAG GGT GAC GGG CGG TGT GT

R-Primer 2: 16SR3 - TTT CAT CTT TCC CTT GCG GTA C

16S rRNA (5' fragment)

F-Primer (3): 16SL3 - AGC AAA GAH YWW ACC TCG TAC CTT TTG CAT

R-Primer (3): 16SAH - ATG TTT TTG ATA AAC AGG CG

16S rRNA (3' fragment)

F-Primer (2): 16SAL - CGC CTG TTT ATC AAA AAC AT

R-Primer (2): 16SBH - CCG GTC TGA ACT CAG ATC ACG T

Appendix B. Voucher specimens and Genbank accession numbers

Voucher specimens are deposited in the herpetological collections of the Museo Regionale di Scienze Naturali, Torino (MRSN), Université d'Antananarivo, Département de Biologie Animale (UADBA), Laboratorio de Biogeografía, Universidad de los Andes, Mérida, Venezuela (ULABG), Zoologisches Forschungsinstitut und Museum A. Koenig, Bonn (ZFMK), Zoologische Staatssammlung München (ZSM). Some UADBA numbers are preliminary fieldnumbers of F. Glaw and M. Vences (UADBA-FG/MV and UADBA-MV), some MRSN numbers are preliminary fieldnumbers of F. Andreone and J. E. Randrianirina (MRSN-FAZC and MRSN-RJS).

Asterisks mark sequences newly obtained in this study.

Mantellidae

Aglyptodactylus madagascariensis: AF249068 (cytochrome b), AF249103 (rhodopsin), AF249166 (tyrosinase), AF249007 (12S and tRNAVal), AY341678* (ZSM 183/2002, Tolagnaro, Madagascar) (16S [5' fragment]), AF249036 (16S [3' fragment]).

Boophis new species (Comoros): AY341733* (cytochrome b), AY341796* (rhodopsin), AY341752* (tyrosinase), AY341610* (12S and tRNAVal), AY341667* (16S [5' fragment]), AY341716* (16S [3' fragment]); all sequences from specimen ZSM 658/2000 (Tsingoni, Mayotte).

Boophis boehmei: AY341798* (rhodopsin), AY341612* (12S and tRNAVal), AY341669* (16S [5' fragment]), AY341717* (16S [3' fragment]); all sequences from specimen UADBA-MV 2001.1205 (Andasibe, Madagascar).

Boophis doulioti: AY341792* (rhodopsin), AY341608* (12S and tRNAVal), AY341663* (16S [5' fragment]), AF215334 (ZFMK 66690, Kirindy, Madagascar) (16S [3' fragment]); all other sequences from specimen ZSM 185/2002 (Nahampoana, Madagascar).

Boophis goudoti: AY341797* (rhodopsin), AY341611* (12S and tRNAVal), AY341668* (16S [5' fragment]), AJ315917 (not preserved; Col des Tapias, Madagascar) (16S [3' fragment]); all other sequences from specimen UADBA-MV 2001.557 (Andringitra, Madagascar).

Boophis idae: AY341795* (rhodopsin), AY341609* (12S and tRNAVal), AY341666* (16S [5' fragment]), AY341715* (16S [3' fragment]); all sequences from specimen ZSM 45/2002 (Andasibe, Madagascar).

Boophis luteus: AY341800* (rhodopsin), AY341614* (12S and tRNAVal), AY341671* (16S [5' fragment]), AJ315916 (UADBA-FG/MV 2000.063, Andasibe, Madagascar) (16S [3' fragment]); all sequences from specimen UADBA-FG/MV 2000.63 (Andasibe, Madagascar).

Boophis marojezensis: AY341803* (rhodopsin), AY341617* (12S and tRNAVal), AY341674* (16S [5' fragment]), AJ315923 (ZSM 326/2000, Vohidrazana, Madagascar) (16S [3' fragment]); all other sequences from specimen ZSM 189/2002 (Vohidrazana, Madagascar).

Boophis microtympanum: AY341799* (rhodopsin), AY341613* (12S and tRNAVal), AY341670* (16S [5' fragment]), AJ315918 (16S [3' fragment]); all sequences from specimen ZSM 393/2000 (Col des Tapias, Madagascar).

Boophis occidentalis: AY341806* (rhodopsin), AY341620* (12S and tRNAVal), AY341677* (16S [5' fragment]), AY341720* (16S [3' fragment]); all sequences from specimen ZSM 44/2002 (Antoetra, Madagascar).

Boophis rappiodes: AY341804* (rhodopsin), AY341618* (12S and tRNAVal), AY341675* (16S [5' fragment]), AJ314815 (16S [3' fragment]); all sequences from specimen ZSM 347/2000 (Andasibe, Madagascar).

Boophis sibilans: AY341801* (rhodopsin), AY341615* (12S and tRNAVal), AY341672* (16S [5' fragment]), AY341718* (16S [3' fragment]); all sequences from specimen ZSM 39/2002 (Andasibe, Madagascar).

Boophis tephraeomystax: AF249070 (cytochrome b), AY341793* (rhodopsin), AF249168 (tyrosinase), AF249009 (12S and tRNAVal), AY341664* (16S [5' fragment]), AJ312116 (16S [3' fragment]); all sequences from specimen UADBA-FG/MV 2000.379 (Sambava, Madagascar).

Boophis vittatus: AY341802* (rhodopsin), AY341616* (12S and tRNAVal), AY341673* (16S [5' fragment]), AY341719* (16S [3' fragment]); all sequences from specimen UADBA-FG/MV 2000.82 (Tsaratanana, Madagascar).

Boophis viridis: AY341805* (rhodopsin), AY341619* (12S and tRNAVal), AY341676* (16S [5' fragment]), AJ314818 (16S [3' fragment]); all sequences from specimen ZSM 338/2000 (Andasibe, Madagascar).

Boophis xerophilus: AY341794* (rhodopsin), AF249008 (12S and tRNAVal), AY341665* (16S [5' fragment]), AF215335 (16S [3' fragment]); all sequences from specimen ZFMK 66705 (Kirindy, Madagascar).

Laliostoma labrosum: AF249096 (cytochrome b), AF249106 (rhodopsin), AF249169 (tyrosinase), AF249010 (12S and tRNAVal), AY341679* (UADBA-MV 2001.289, Ankarafantsika, Madagascar) (16S [5' fragment]), AF249037 (16S [3' fragment]).

Mantella laevigata: AY263277 (rhodopsin), AY341607* (12S and tRNAVal), AJ438538 (16S [5' fragment]), AF215279 (16S [3' fragment]).

Mantella madagascariensis: AF249076 (cytochrome b), AF249101 (rhodopsin), AF249164 (tyrosinase), AF249005 (12S and tRNAVal), AJ438892 (16S [5' fragment]), AF249049 (16S [3' fragment]).

Mantidactylus new species (Comoros): AY341731* (cytochrome b), AY323742* (rhodopsin), AY341750* (tyrosinase), AY341585* (12S and tRNAVal), AY341639* (16S [5' fragment]), AY330888* (16S [3' fragment]); all sequences from specimen ZSM 652/2000 (Mont Combani, Mayotte).

Mantidactylus albolineatus: AY341766* (rhodopsin), AY341580* (12S and tRNAVal), AY341635* (16S [5' fragment]), AY341701* (16S [3' fragment]); all sequences from specimen ZSM 250/2002 (Andasibe, Madagascar).

Mantidactylus ambreensis: AY341788* (rhodopsin), AY341603* (12S and tRNAVal), AY341659* (16S [5' fragment]), AY324822* (ZSM 492/2000, Montagne d'Ambre) (16S [3' fragment]); all other sequences from specimen ZSM 634/2001 (Tsaratanana, Madagascar).

Mantidactylus asper: AY341783* (rhodopsin), AY341598* (12S and tRNAVal), AY341653* (16S [5' fragment]), AJ314802 (16S [3' fragment]); all sequences from specimen UADBA- FG/MV 2000.17 (Mandraka, Madagascar).

Mantidactylus biporus: AY341784* (rhodopsin), AY341599* (12S and tRNAVal), AY341655* (16S [5' fragment]), AF215322 (ZFMK 70481, Masoala) (16S [3' fragment]); all other sequences from specimen ZSM 122/2002 (Andranofotsy, Madagascar).

Mantidactylus blommersae: AY341770* (rhodopsin), AY341584* (12S and tRNAVal), AY341638* (16S [5' fragment]), AF317688 (16S [3' fragment]); all sequences from specimen UADBA-FG/MV 2000.65 (Andasibe, Madagascar).

Mantidactylus charlotteae: AY341790* (rhodopsin), AY341605* (12S and tRNAVal), AY341661* (16S [5' fragment]), AY341713* (16S [3' fragment]); all sequences from specimen ZSM 127/2002 (Andranofotsy, Madagascar).

Mantidactylus depressiceps: AY341775* (rhodopsin), AY341590* (12S and tRNAVal), AY341645* (16S [5' fragment]), AF215326 (ZFMK 60131, Andasibe) (16S [3' fragment]); all other sequences from specimen ZSM 688/2001 (Andasibe, Madagascar).

Mantidactylus domerguei: AY341768* (rhodopsin), AY341582* (12S and tRNAVal), AY341636* (16S [5' fragment]), AF317689 (16S [3' fragment]); all sequences from specimen ZSM 353/2000 (Mantasoa, Madagascar).

Mantidactylus massorum: AY341776* (rhodopsin), AY341591* (12S and tRNAVal), AY341646* (16S [5' fragment]), AY341705* (16S [3' fragment]); all sequences from specimen MRSN-FAZC 6737 (Ambolokopatrika, Madagascar).

Mantidactylus grandidieri: AY341789* (rhodopsin), AY341604* (12S and tRNAVal), AY341660* (16S [5' fragment]), AY341712* (16S [3' fragment]); all sequences from specimen UADBA-MV 2001.1201 (Andasibe, Madagascar).

Mantidactylus grandisonae: AY341771* (rhodopsin), AY341640* (16S [5' fragment]), AF215315 (16S [3' fragment]); all sequences from specimen ZFMK 66669 (Ambato, Madagascar).

Mantidactylus granulatus: AY341779* (rhodopsin), AY341594* (12S and tRNAVal), AY341649* (16S [5' fragment]), AJ314794 (ZSM 645/2001, Tsaratanana) (16S [3' fragment]); all other sequences from specimen MRSN-FAZC 8011 (Nosy Be, Madagascar).

Mantidactylus horridus: AY341781* (rhodopsin), AY341596* (12S and tRNAVal), AY341651* (16S [5' fragment]), AY341708* (16S [3' fragment]); all sequences from specimen UADBA 10002 (Tsaratanana, Madagascar).

Mantidactylus kely: AY341769* (rhodopsin), AY341583* (12S and tRNAVal), AY341637* (16S [5' fragment]), AF317690 (16S [3' fragment]); all sequences from specimen ZSM 363/2000 (Ambatolampy, Madagascar).

Mantidactylus liber: AY341774* (rhodopsin), AY341589* (12S and tRNAVal), AY341644* (16S [5' fragment]), AJ314801 (16S [3' fragment]); all sequences from specimen ZSM 491/2000 (Montagne d'Ambre, Madagascar).

Mantidactylus lugubris: AY341785* (rhodopsin), AY341600* (12S and tRNAVal), AY341656* (16S [5' fragment]), AY341710* (16S [3' fragment]); all sequences from specimen ZSM 166/2002 (Mantady, Madagascar).

Mantidactylus new species (aff. lugubris): AY341786* (rhodopsin), AY341601* (12S and tRNAVal), AY341657* (16S [5' fragment]), AY341711* (16S [3' fragment]); all sequences from specimen ZSM 171/2002 (Mantady, Madagascar).

Mantidactylus madinika: AY341772* (rhodopsin), AY341587* (12S and tRNAVal), AY341642* (16S [5' fragment]), AY341703* (16S [3' fragment]); all sequences from one paratype specimen from Antsirasira, Madagascar.

Mantidactylus femoralis: AY341787* (rhodopsin), AY341602* (12S and tRNAVal), AY341658* (16S [5' fragment]), AY324812* (16S [3' fragment]); all sequences from specimen UADBA-MV 2001.1277 (Andasibe, Madagascar).

Mantidactylus opiparis: AY341791* (rhodopsin), AY341606* (12S and tRNAVal), AY341662* (16S [5' fragment]), AY341714* (16S [3' fragment]); all sequences from specimen UADBA-MV 2001.1069 (Marolambo region, Madagascar).

Mantidactylus peraccae: AY341777* (rhodopsin), AY341592* (12S and tRNAVal), AY341647* (16S [5' fragment]), AY341706* (16S [3' fragment]); all sequences from specimen MRSN-RJS 109 (Tsaratanana, Madagascar).

Mantidactylus redimitus: AY341778* (rhodopsin), AY341593* (12S and tRNAVal), AY341648* (16S [5' fragment]), AY341707* (16S [3' fragment]); all sequences from specimen ZSM 152/2002 (Vohidrazana, Madagascar).

Mantidactylus sculpturatus: AY341782* (rhodopsin), AY341597* (12S and tRNAVal), AY341652* (16S [5' fragment]), AY341709* (16S [3' fragment]); all sequences from specimen ZSM 95/2002 (Mantady, Madagascar).

Mantidactylus striatus: AY341780* (rhodopsin), AY341595* (12S and tRNAVal), AY341650* (16S [5' fragment]), AJ314796* (16S [3' fragment]).

Mantidactylus aff. ulcerosus: AF249102* (rhodopsin), AF249006* (12S and tRNAVal), AY341654* (16S [5' fragment]), AF215319 (16S [3' fragment]); all sequences from specimen ZFMK 66659 (Ambato, Madagascar).

Mantidactylus wittei: AY341732* (cytochrome b), AY323743* (rhodopsin), AY341751* (tyrosinase), AY341586* (12S and tRNAVal), AY341641* (16S [5' fragment]), AF317691 (UADBA-FG/MV 2000.123, Ambanja) (16S [3' fragment]); all other sequences from specimen ZSM 405/2000 (Benavony, Madagascar).

Ranidae

Hoplobatrachus crassus: AF249090 (cytochrome b), AF249109 (rhodopsin), AF249172 (tyrosinase), AF249013 (12S and tRNAVal), AY341688* (16S [5' fragment]), AY014375 (16S [3' fragment]).

Hoplobatrachus occipitalis: AJ564733* (cytochrome b), AJ564730* (rhodopsin), AJ564729* (tyrosinase), AJ564734* (12S and tRNAVal), AY341689* (16S [5' fragment]), AY014373 (16S [3' fragment]); all sequences from specimen ZFMK-WB 02 (Mauritania).

Indirana cf. leptodactyla: AF215392 (16S [5' fragment]), AY341686* (16S [3' fragment]); sequences from specimen ZFMK uncatalogued (Ooty, India).

Indirana sp. (aff. leptodactyla): AF249080 (cytochrome b), AF249123 (rhodopsin), AF249186 (tyrosinase), AF249027 (12S and tRNAVal).

Nyctibatrachus major: AF249084 (cytochrome b), AF249113 (rhodopsin), AF249176 (tyrosinase), AF249017 (12S and tRNAVal), AY341687* (16S [5' fragment]), AF215397 (16S [3' fragment]).

Petropedetes parkeri: AY341813* (rhodopsin), AY341757* (tyrosinase), AY341628* (12S and tRNAVal), AY341694* (16S [5' fragment]), AY341724* (16S [3' fragment]); all sequences from a specimen from Nlonako (Cameroon).

Petropedetes sp. (aff. parkeri): AY341738* (cytochrome b); sequence from a specimen from Cameroon.

Ptychadena mascareniensis: AY341734* (cytochrome b), AY341809* (rhodopsin), AY341753* (tyrosinase), AY341624* (12S and tRNAVal), AY341690* (16S [5' fragment]); all sequences from specimen ZSM 258/2002 (Nahampoana, Madagascar ).

Rana temporalis: AF249083 (cytochrome b), AF249118 (rhodopsin), AF249181 (tyrosinase), AF249022 (12S and tRNAVal), AY341683* (16S [5' fragment]), AF215390 (16S [3' fragment]).

Rana temporaria: AF249078 (cytochrome b), AF249119 (rhodopsin), AF249182 (tyrosinase), AF249023 (12S and tRNAVal), AY341684* (16S [5' fragment]), AF249048 (16S [3' fragment]).

Rana cretensis: AY148010 (rhodopsin).

Rana cerigensis: AY148009 (rhodopsin).

Rhacophoridae

Polypedates cruciger: AF249089 (cytochrome b), AF249124 (rhodopsin), AF249187 (tyrosinase), AF249028 (12S and tRNAVal), AY341685* (16S [5' fragment]), AF215357 (16S [3' fragment]).

Astylosternidae

Astylosternus diadematus: AY341735* (cytochrome b), AY341810* (rhodopsin), AY341754* (tyrosinase), AY341625* (12S and tRNAVal), AY341691* (16S [5' fragment]), AY341723* (16S [3' fragment]); all sequences from a specimen from Nlonako (Cameroon).

Arthroleptidae

Arthroleptis variabilis: AY341737* (cytochrome b), AY341812* (rhodopsin), AY341756* (tyrosinase), AY341627* (12S and tRNAVal), AY341693* (16S [5' fragment]), AF124107 (ZFMK 68794, Cameroon) (16S [3' fragment]); all other sequences from a specimen from Nlonako (Cameroon).

Hyperoliidae

Leptopelis natalensis: AY341736* (cytochrome b), AY341811* (rhodopsin), AY341755* (tyrosinase), AY341626* (12S and tRNAVal), AY341692* (16S [5' fragment]), AF215448 (16S [3' fragment]); all sequences from specimen ZFMK 68785 (Mtunzini, South Africa).

Hyperolius cf. viridiflavus: AF249066 (cytochrome b), AF249098 (rhodopsin), AF249161 (tyrosinase), AF249002 (12S and tRNAVal).

Hyperolius viridiflavus: AY341695* (16S [5' fragment]), AF215440 (16S [3' fragment]); sequences from specimen ZFMK 66726 (Barberton, South Africa).

Tachycnemis seychellensis: AY341739* (cytochrome b), AY341814* (rhodopsin), AY341758* (tyrosinase), AY341629* (12S and tRNAVal), AY341696* (16S [5' fragment]), AF215451 (16S [3' fragment]).

Heterixalus tricolor: AY341740* (cytochrome b), AY323741* (rhodopsin), AY341759* (tyrosinase), AY341630* (12S and tRNAVal), AY341697* (16S [5' fragment]), AY341725* (16S [3' fragment]); all sequences from specimen ZSM 700/2001 (Ankarafantsika, Madagascar).

Bufonidae

Bufo melanostictus: AF249082 (cytochrome b), AF249097 (rhodopsin), AF249001 (12S and tRNAVal), AF249061 (16S [3' fragment]).

Bufo asper: AY263257 (16S [5' fragment]).

Leptodactylidae

Leptodactylus fuscus: AY341741* (cytochrome b), AY323746* (rhodopsin), AY341760* (tyrosinase), AY341631* (12S and tRNAVal), AY263262* (16S [5' fragment]), AY263226* (16S [3' fragment]); all sequences from specimen ULABG 4591 (Canaima, Venezuela).

Sooglossidae

Nesomantis thomasseti: AY341742* (cytochrome b), AY323744* (rhodopsin), AY341761* (tyrosinase), AY341632* (12S and tRNAVal), AY341698* (16S [5' fragment]), AY330889* (16S [3' fragment]).

Pipidae

Pipa parva: AY341743* (cytochrome b), AY323734* (rhodopsin), AY341762* (tyrosinase), AY341633* (12S and tRNAVal), AY341699* (16S [5' fragment]), AY333690* (16S [3' fragment]).

Xenopus laevis: M10217 (cytochrome b), S62229 (rhodopsin), AY341764* (tyrosinase), M10217 (12S and tRNAVal), M10217 (16S [5' fragment]), AY341727* (16S [3' fragment]).

Hymenochirus boettgeri: AY341744* (cytochrome b), AY323735* (rhodopsin), AY341763* (tyrosinase), AY341634* (12S and tRNAVal), AY341700* (16S [5' fragment]), AY341726* (16S [3' fragment]).

Pelobatidae

Pelobates cultripes: AY323736* (rhodopsin).

Pelobates varaldi: AY341815* (rhodopsin).

Discoglossidae

Alytes muletensis: AY341728* (cytochrome b), AY323731* (rhodopsin), AY341747* (tyrosinase), AY341621* (12S and tRNAVal), AY341680* (16S [5' fragment]), AF224729 (16S [3' fragment]); all sequences from a specimen preserved in the ZFMK (Mallorca, Spain).

Alytes maurus: AY341816* (rhodopsin).

Alytes dickhillenii: AY341817* (rhodopsin).

Urodela

Ambystoma mexicanum: AY341745* (cytochrome b), U36574 (rhodopsin), Y10947 (12S and tRNAVal), Y10947 (16S [5' fragment]), Y10947 (16S [3' fragment]).

Hynobius kimurae: AY341746 (cytochrome b), AY341765* (tyrosinase).

Appendix C. Substitution models used in Maximum Likelihood Phylogenetic analysis

We tested the goodness-of-fit of nested substitution models for homogeneous data partitions of ingroup taxa by a hierarchical likelihood ratio test (HLRT). Modeltest (4) version 3.06 was used to calculate the test statistic δ = 2 log Λ with Λ being the ratio of the likelihood of the null model divided by the likelihood of the alternative model (5). The best fitting model was used for Maximum Likelihood phylogenetic analyses using PAUP* (6).

Before applying the HLRT and any phylogenetic analysis, we excluded all gapped and hypervariable regions of the rRNA and tRNA genes. Rather than trying to fit these regions (which mainly correspond to loops in the tertiary structure of the rRNA molecules) into secondary structure models (7), we preferred to exclude all sections in which homology was not immediately obvious, as well as all gapped positions. Additionally, we excluded the third positions of cytochrome b which are known to be fully saturated at the level of anuran families (8).

The following substitution models were selected by the hierarchical likelihood ratio test (HLRT) as implemented in Modeltest:

For the among-Mantellidae dataset of the concatenated rhodopsin, 12S rRNA, 16SrRNA and tRNAVal sequences, a GTR+I+G substitution model (-lnL = 26025.7305) with empirical base frequencies (freqA = 0.3551; freqC = 0.2199; freqG = 0.1644; freqT = 0.2607) and substitution rates ([A-C] = 3.7878; [A-G] = 10.5790; [A-T] = 5.7768; [C-G] = 1.1797; [C-T] = 29.2325; [G- T] = 1), a proportion of invariable sites of 0.4042 and a gamma distribution shape parameter of 0.6582.

For the higher-level relationship data set of the concatenated rhodopsin, tyrosinase, cytochrome b, 12SrRNA, 16S rRNA, and tRNAVal sequences, a TrN+I+G substitution model (-lnL = 30463.0156), with empirical base frequencies (freqA = 0.3371; freqC = 0.2527; freqG = 0.1497; freqT = 0.2606) and substitution rates ([A-G] = 3.3874; [C-T] = 5.2170; all other rates = 1), a proportion of invariable sites of 0.2851, and a gamma distribution shape parameter of 0.6848.

For the higher-level relationship data set of nuclear genes only (rhodopsin, tyrosinase), a HKY+I+G substitution model (-lnL = 8573.2109) with empirical base frequencies (freqA = 0.2261; freqC = 0.3242; freqG = 0.2048; freqT = 0.2449), a transition/transversion ratio of 2.1689, a proportion of invariable sites of 0.3788, and a gamma distribution shape parameter of 1.25.

For the higher-level relationship data set of rRNA and tRNA genes only (12S rRNA, 16S rRNA, tRNAVal), a GTR+I+G substitution model (-lnL = 17708.6992) with empirical base frequencies (freqA = 0.3763; freqC = 0.2075; freqG = 0.1800; freqT = 0.2362) and substitution rates ([A-C] = 3.8266; [A-G] = 7.5824; [A-T] = 5.2028; [C-G] = 0.6641; [C-T] = 24.8576; [G-T] = 1), a proportion of invariable sites of 0.1657, and a gamma distribution shape parameter of 0.5400.

Appendix D. Details of Bayesian phylogenetic analysis

Bayesian phylogenetic analyses were performed using the program MrBayes, version 2.01 (9). The analysis consisted of Maximum Likelihood (ML) comparisons of trees in which the tree topology and ML parameters were permuted using a Markov chain Monte Carlo method with Metropolis-coupling (MCMCMC) (9), and sampled periodically according to the Metropolis- Hastings algorithm. Because Modeltest (4) suggested complex substitution models (GTR+I+G; see previous section), we set the ML parameters in MrBayes as follows: "lset nst = 6" (the GTR model), "rates = invgamma" (site-specific rate variation, with some invariant sites and other sites with a gamma distribution), and "basefreq = estimate" (proportion of different base categories estimated from the data).

All our analyses employed one cold chain and three incrementally heated chains. We run 500,000 generations in the higher-level phylogeny (Fig. 5) and 300,000 generations in the mantellid phylogeny (fig. 4). Trees were sampled every ten generations. The initial set of generations needed before convergence on stable likelihood values (burnin) was set at 6-7% based on own empirical evaluation.

Appendix E. Separate analysis of gene fragments

The following figures show the results of additional analyses of the two data sets. For the Mantellidae data set, we present the maximum likelihood (ML) phylogram with maximum parsimony (MP) bootstrap values. For the higher-level relationship data set, we show (a) a ML analysis of nuclear genes (rhodopsin and tyrosinase) at the amino acid level, (b) a ML analysis of nuclear genes only at the amino acid level, but using Alytes muletensis (Discoglossidae) as outgroup instead of a salamander, ( c) a ML analysis of nuclear genes only at the nucleotide level, and (d) a ML analysis based on ribosomal genes only (12SrRNA, 16SrRNA, tRNAVal). Bootstrap values are shown for ML, MP and Neighbor-joining (NJ).

Fig. 4. ML phylogram showing relationships among the Mantellidae, obtained by heuristic searches in PAUP* using settings suggested by Modeltest. The numbers are bootstrap values in percent under MP (2000 replicates).

Fig. 5. ML phylogram showing higher-level relationships among Gondwanan frogs. Based on amino acid sequences of nuclear genes (rhodopsin and tyrosinase; VT substitution model (10)). The numbers are quartet puzzling values in percent under ML (10,000 puzzling steps, obtained using Tree-Puzzle (11)), bootstrap values under MP (2000 replicates; obtained using PAUP*, with 10 random addition sequence replicates) and NJ (obtained using Mega (12), under minimum evolution criterion using an empirically determined gamma distribution shape parameter with eight rate categories).

77 Mantidactylus new species Comoros ML 99 NJ 95 Mantidactylus wittei MP Aglyptodactylus madagascariensis 67 Boophis tephraeomystax 98 95 Boophis new species Comoros

0.1 substitutions/site Laliostoma labrosum 82 80 Rana galamensis 60 76 Rana temporalis 69 87 41 Hoplobatrachus crassus 100 100 Hoplobatrachus occipitalis 96 90 Chiromantis rufescens 93 Polypedates cruciger ---- 61 Indirana cf leptodactyla 49 Nyctibatrachus major 54 Ptychadena mascareniensis 39 76 48 Petropedetes parkeri 96 93 54 Tachycnemis seychellensis 83 87 41 100 Heterixalus tricolor 100 ---- Hyperolius viridiflavus 55 69 Leptopelis natalensis 30 79 72 ---- Astylosternus diadematus 46 Arthroleptis variabilis 40 Leptodactylus fuscus 87 59 Nesomantis thomasseti 65 Pipa parva 71 94 Hymenochirus boettgeri 89 Xenopus laevis

Alytes muletensis

Ambyostoma mexicanum

Fig. 6. ML phylogram showing higher-level relationships among Gondwanan frogs. The numbers are bootstrap values in percent under ML, MP, and NJ. Based on amino acid sequences of nuclear genes (rhodopsin and tyrosinase). Because of the high divergence of the salamander sequences, this separate tree was calculated using the basal discoglossid frog Alytes muletensis as outgroup. See legend of Fig. 5 for further details of analysis.

76 52 Mantidactylus new species Comoros ML 99 NJ ------94 Mantidactylus wittei MP Aglyptodactylus madagascariensis 62 Boophis tephraeomystax 41 32 ---- 45 Boophis new species Comoros ---- Laliostoma labrosum

0.1 substitutions/site Nyctibatrachus major 50 Ptychadena mascareniensis 62 40 32 56 Petropedetes parkeri 45 Indirana cf leptodactyla ---- 95 49 Chiromantis rufescens 90 43 92 Polypedates cruciger 87 Hoplobatrachus crassus 100 76 83 100 Hoplobatrachus occipitalis 67 79 74 47 65 Rana galamensis 96 92 Rana temporalis 94 56 Tachycnemis seychellensis 88 45 100 Heterixalus tricolor 55 100 ---- Hyperolius viridiflavus 67 29 32 Leptopelis natalensis ------81 ---- 32 Astylosternus diadematus ---- 66 Arthroleptis variabilis

Leptodactylus fuscus

Nesomantis thomasseti ---- Pipa parva 70 56 94 70 Hymenochirus boettgeri 89 Xenopus laevis

Alytes muletensis

Fig. 7. ML phylogram showing higher-level relationships among Gondwanan frogs. The numbers are bootstrap values in percent under ML, MP and NJ. Based on nucleotide sequences of nuclear genes (rhodopsin and tyrosinase). See legend of Fig. 5 for further details of analysis.

84 Chiromantis rufescens 100 ML 98 Polypedates cruciger NJ 84 MP Mantidactylus new species Comoros 100 100 Mantidactylus wittei

Aglyptodactylus madagascariensis 88 Boophis tephraeomystax 100 0.05 substitutions/site 100 Boophis new species Comoros

Laliostoma labrosum

83 Rana galamensis 100 100 Rana temporalis 97 Hoplobatrachus crassus 100 40 100 Hoplobatrachus occipitalis 99 95 Nyctibatrachus major 59 Indirana cf leptodactyla 93 81 Petropedetes parkeri 63 100 Ptychadena mascareniensis 100 83 Astylosternus diadematus 94 59 Leptopelis natalensis 62 ---- Arthroleptis variabilis 72 100 51 94 92 Hyperolius viridiflavus 100 63 100 Tachycnemis seychellensis 35 86 79 100 Heterixalus tricolor 98 Leptodactylus fuscus

Nesomantis thomasseti 78 Pipa parva 72 22 100 61 Hymenochirus boettgeri 100 Xenopus laevis

Alytes muletensis

Fig. 8. ML phylogram showing higher-level relationships among Gondwanan frogs. Obtained by heuristic searches in PAUP* with TBR branch-swapping and 10 random sequence addition replicates, under the substitution model proposed by Modeltest. The numbers are bootstrap values in percent under ML (100 replicates), Maximum Parsimony (2000 replicates), and Neighbor-joining (2000 replicates). Based on nucleotide sequences of ribosomal genes

(12SrRNA, 16SrRNA, tRNAVal) only. Although the ML tree did not place Nesomantis (Sooglossidae) as sister group of all Neobatrachia, such a topology was suggested by the MP and NJ bootstrap consensus trees (66% and 63% support), in agreement with the trees based on nuclear genes (see Figs. 5-7).

Appendix F. Testing alternative topologies with Shimodaira-Hasegawa tests

Non-parametric likelihood ratio tests (Shimodaira-Hasegawa tests - SH tests) (13) as implemented in PAUP* (6), were used to test alternative topologies (chosen a priori) in the phylogenetic trees obtained by Maximum Likelihood searches. This is the only statistically valid method currently available for multiple topology testing (14). We run SH tests with 2000 bootstrap replicates and full optimization settings. The following tables show the topologies tested, their likelihood scores, the difference in likelihood score as compared to the best tree, and the significance of this difference.

In the mantellid phylogeny, we tested alternative positions of the two Comoroan species, keeping the overall topology unchanged. The results, summarized in Table 1, show that the general position of these taxa is statistically significant. The only topologies that could not be excluded were (a) permutations of the position of Mantidactylus sp. (Comoros) relative to M. wittei, M. domerguei and M. blommersae, and (b) of Boophis sp. (Comoros) relative to B. tephraeomystax, B. doulioti and B. xerophilus. Considering any of these species other than M. wittei and B. tephraeomystax as sister taxa of the Comoroan species would result in higher genetic divergences of the Comoroan taxa, thus reinforcing the molecular clock calculations performed by suggesting even younger origins of the lineages studied. In contrast, hypotheses placing the Comoroan taxa as sister groups to each other, or as basal to their respective lineages (subgenera or species groups) could be significantly rejected. This provides evidence that the Comoroan taxa are deeply nested within their clades, and strongly confirms their double independent origin. Although the Comoroan Mantidactylus had previously been included in Mantidactylus granulatus, a relationship to this species or its relatives (M. redimitus, M. sculpturatus) could also be excluded with high significance. In the taxa set used to assess higher-level relationships, the SH-tests significantly excluded relationships between the two Seychellean taxa Tachycnemis and Nesomantis, confirming the independent origin of the two Seychellean frog lineages. In contrast, the arrangement of the different mantellid subfamilies was not sufficiently clarified, and also several alternative positions of Nesomantis could not be significantly excluded, indicating that our data set is insufficient for resolving satisfyingly the deep splits among the main frog lineages.

Table 1. Results of SH-tests in the Mantellidae data set. M. sp. and B. sp. refer to the Mantidactylus and Boophis species from Mayotte (Comoros). Significances of P<0.05 are marked with an asterisk.

Tree Description of ree permutation Likelihood (-lnL) Diff (-lnL) Significance A0 Best ML tree 26013.35215 ------A1 M. sp. sister to M. domerguei / M. blommersae 26042.78229 29.43014 0.5185 A2 M. sp. sister to M. wittei / M. domerguei / M. blommersae 26042.78229 29.43014 0.5185 A3 M. sp. sister to M. domerguei 26074.46334 61.11119 0.0730 A4 M. sp. sister to M. blommersae 26074.84296 61.49082 0.0695 A5 M. sp. sister to M. grandisonae / M. kely / M. sarotra 26090.12128 76.76914 0.0160* A6 M. sp. sister to M. grandisonae 26108.24787 94.89572 0.0045* A7 M. sp. sister to M. kely / M. sarotra 26108.24787 94.89572 0.0045* A8 M. sp. sister to M. kely 26187.82919 174.47704 0.0000* A9 M. sp. sister to M. sarotra 26187.82919 174.47704 0.0000* A10 M. sp. sister to all Blommersia 26090.12128 76.76914 0.0160* A11 M. sp. sister to M. madinika / Mantella spp. 26164.94468 151.59254 0.0000* A12 M. sp. sister to M. madinika 26205.35170 191.99955 0.0000* A13 M. sp. sister to species of Guibemantis / Pandanusicola 26133.00009 119.64794 0.0005* A14 M. sp. sister to species of Guibemantis / Pandanusicola 26188.96063 175.60848 0.0000* A15 M. sp. sister to species of Guibemantis / Pandanusicola 26188.96063 175.60848 0.0000* A16 M. sp. sister to M. granulatus 26381.78107 368.42892 0.0000* A17 M. sp. sister to M. redimitus 26381.78107 368.42892 0.0000* A18 M. sp. sister to M. granulatus / M. redimitus 26346.10407 332.75192 0.0000* A19 M. sp. sister to M. sculpturatus / M. granulatus / M.redimitus 26338.20727 324.85512 0.0000* A20 B. sp. sister to M. sp. 26465.21993 451.86778 0.0000* A21 M. sp. sister to B. sp. 26443.05343 429.70129 0.0000* A22 B. sp. sister to B. tephraeomystax / B.doulioti 26014.00790 0.65576 0.9875 A23 B. sp. sister to B. tephraeomystax 26016.30986 2.95772 0.9785 A24 B. sp. sister to B. xerophilus 26068.06433 54.71218 0.1380 A25 B. sp. sister to B. doulioti / B. tephraeomystax / B. xerophilus 26069.01388 55.66174 0.1245 A26 B. sp. sister to B. idae 26117.88218 104.53003 0.0035* A27 B. sp. sister to all pond breeding Boophis 26117.88218 104.53003 0.0035* A28 B. sp. sister to all Boophis 26173.48306 160.13091 0.0000* A29 B. sp. sister to all brook breeding Boophis 26173.48306 160.13091 0.0000*

Table 2. Results of SH-tests in the higher-level relationship data set. Significances of P<0.05 are marked with an asterisk.

Tree Description of ree permutation Likelihood (-lnL) Diff (-lnL) Significance B0 Best ML tree 30444.99194 ------B1 Different position of Nesomantis 30447.52743 2.53548 0.940 B2 Different position of Nesomantis 30508.07683 63.08489 0.085 B3 Different position of Nesomantis 30510.36233 65.37039 0.077 B4 Different position of Nesomantis 30516.91704 71.92510 0.060 B5 Nesomantis sister to Ranoidea 30449.36063 4.36869 0.935 B6 Nesomantis sister to Ranidei 30509.67657 64.68463 0.044* B7 Nesomantis sister to Arthroleptoidei 30508.79564 63.80370 0.050 B8 Nesomantis sister to Tachycnemis 30644.78662 199.79468 0.000* B9 Tachycnemis sister to Nesomantis 31004.34740 559.35546 0.000* B10 Rhacophoridae sister of Mantellidae 30451.63068 6.63874 0.847 B11 Rhacophoridae sister of Mantellidae 30450.20593 5.21398 0.859 B12 Different arrangement of mantellid subfamilies 30448.63226 3.64031 0.942 B13 Different arrangement of mantellid subfamilies 30452.15834 7.16640 0.891 B14 Different arrangement of mantellid subfamilies 30489.00288 44.01094 0.217

Table 3. (next page). Topologies of trees included in Shimodaira-Hasegawa non-parametric likelihood ratio tests (dataset: Mantellidae). Taxa: 1, Polypedates cruciger; 2, Mantidactylus depressiceps; 3, Mantidactylus albolineatus; 4, Mantidactylus liber; 5, Mantidactylus domerguei; 6, Mantidactylus blommersae; 7, Mantidactylus new species Comoros; 8, Mantidactylus wittei; 9, Mantidactylus grandisonae; 10, Mantidactylus kely; 11, Mantidactylus sarotra; 12, Mantidactylus madinika; 13, Mantella madagascariensis; 14, Mantella laevigata; 15, Mantidactylus fimbriatus; 16, Mantidactylus peraccae; 17, Mantidactylus sculpturatus; 18, Mantidactylus redimitus; 19, Mantidactylus granulatus; 20, Mantidactylus asper; 21, Mantidactylus striatus; 22, Mantidactylus horridus; 23, Mantidactylus grandidieri; 24, Mantidactylus aff ulcerosus; 25, Mantidactylus biporus; 26, Mantidactylus charlotteae; 27, Mantidactylus opiparis; 28, Mantidactylus lugubris; 29, Mantidactylus sp aff lugubris; 30, Mantidactylus mocquardi; 31, Mantidactylus ambreensis; 32, Aglyptodactylus madagascariensis; 33, Laliostoma labrosum; 34, Boophis idae; 35, Boophis xerophilus; 36, Boophis tephraeomystax; 37, Boophis doulioti; 38, Boophis new species Comoros; 39, Boophis microtympanum; 40, Boophis occidentalis; 41, Boophis goudoti; 42, Boophis boehmei; 43, Boophis luteus; 44, Boophis sibilans; 45, Boophis rappiodes; 46, Boophis viridis; 47, Boophis vittatus; 48, Boophis marojezensis; 49, Rana temporaria; 50, Rana temporalis.

Tree Topology

(1, ( ( ( ( ( (2, (3,4)), ( ( (5,6), (7,8)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A0 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (7, (5,6))), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A1 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (7, (8, (5,6))), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A2 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (6, (5,7))), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A3 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (5, (6,7))), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A4 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (5,6)), (7, (9, (10,11))))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A5 (47,48)))), (49,50)) (1, ( ( ( ( ( (2, (3,4)), ( (8, (5,6)), ( (7,9), (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A6 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (5,6)), (9, (7, (10,11))))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A7 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (5,6)), (9, (11, (7,10))))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A8 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (5,6)), (9, (10, (7,11))))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A9 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), (7, ( (8, (5,6)), (9, (10,11))))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A10 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (5,6)), (9, (10,11)))), (7, (12, (13,14)))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A11 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (5,6)), (9, (10,11)))), ( (7,12), (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A12 (47,48)))), (49,50))

(1, ( ( ( ( ( (7, (2, (3,4))), ( (8, (5,6)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A13 (47,48)))), (49,50))

(1, ( ( ( ( ( ( (2,7), (3,4)), ( (8, (5,6)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A14 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (7, (3,4))), ( (8, (5,6)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A15 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (5,6)), (9, (10,11)))), (12, (13,14))), ( ( ( (17, (18, (7,19))), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))), (15,16))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A16 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (5,6)), (9, (10,11)))), (12, (13,14))), ( ( ( (17, (19, (7,18))), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))), (15,16))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A17 (47,48)))), (49,50)) (1, ( ( ( ( ( (2, (3,4)), ( (8, (5,6)), (9, (10,11)))), (12, (13,14))), ( ( ( (17, (7, (18,19))), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))), (15,16))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A18 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (5,6)), (9, (10,11)))), (12, (13,14))), ( ( ( (7, (17, (18,19))), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))), (15,16))), (32,33)), ( (34, (35, (36, (37,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A19 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( ( (5,6), (8, (7,38))), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36,37))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A20 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( (8, (5,6)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36, (37, (7,38))))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A21 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( ( (5,6), (7,8)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (38, (36,37)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A22 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( ( (5,6), (7,8)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (37, (36,38)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A23 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( ( (5,6), (7,8)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, ( (35,38), (36,37))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A24 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( ( (5,6), (7,8)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (38, (35, (36,37)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A25 (47,48)))), (49,50)) (1, ( ( ( ( ( (2, (3,4)), ( ( (5,6), (7,8)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( ( (34,38), (35, (36,37))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A26 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( ( (5,6), (7,8)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (38, (34, (35, (36,37)))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A27 (47,48)))), (49,50))

(1, ( ( ( ( ( (2, (3,4)), ( ( (5,6), (7,8)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), (38, ( (34, (35, (36,37))), ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A28 (47,48))))), (49,50)) (1, ( ( ( ( ( (2, (3,4)), ( ( (5,6), (7,8)), (9, (10,11)))), (12, (13,14))), ( (15,16), ( ( (17, (18,19)), (20, (21,22))), (23, ( ( ( (24,25), (26,27)), (28,29)), (30,31)))))), (32,33)), ( (34, (35, (36,37))), (38, ( ( ( (39, (40, (41,42))), (43,44)), (45,46)), A29 (47,48))))), (49,50))

Table 4. Topologies of trees included in Shimodaira-Hasegawa non-parametric likelihood ratio tests (dataset: higher-level rfelationships). Taxa: 1, Alytes muletensis; 2, Chiromantis rufescens; 3, Rana galamensis; 4, Mantidactylus new species Comoros; 5, Mantidactylus wittei; 6, Boophis tephraeomystax; 7, Boophis new species Comoros; 8, Aglyptodactylus madagascariensis; 9, Laliostoma labrosum; 10, Rana temporalis; 11, Polypedates cruciger; 12, Indirana cf leptodactyla; 13, Nyctibatrachus major; 14, Hoplobatrachus crassus; 15, Hoplobatrachus occipitalis; 16, Ptychadena mascareniensis; 17, Astylosternus diadematus; 18, Leptopelis natalensis; 19, Arthroleptis variabilis; 20, Petropedetes parkeri; 21, Hyperolius viridiflavus; 22, Tachycnemis seychellensis; 23, Heterixalus tricolor; 24, Bufo melanostictus; 25, Leptodactylus fuscus; 26, Nesomantis thomasseti; 27, Pipa parva; 28, Hymenochirus boettgeri; 29, Xenopus laevis; 30, Ambystoma mexicanum

Tree Topology B0 ( (1, ( ( ( ( ( ( ( ( ( (2,11),13), (3,10)), ( (4,5), ( (6,7), (8,9)))), (12, (16,20))), (14,15)), ( ( (17,18),19), (21, (22,23)))), (24,25)),26), ( (27,28),29))),30) B1 (30, (1, ( ( ( ( ( ( ( (13, (2,11)), (3,10)), ( (4,5), ( (6,7), (8,9)))), (12, (16,20))), (14,15)), ( (19, (17,18)), (21, (22,23)))), (26, (24,25))), (29, (27,28))))) B2 (30, (1, (26, ( ( ( ( ( ( ( (13, (2,11)), (3,10)), ( (4,5), ( (6,7), (8,9)))), (12, (16,20))), (14,15)), ( (19, (17,18)), (21, (22,23)))), (24,25)), (29, (27,28)))))) B3 (30, (1, ( ( ( ( ( ( ( (13, (2,11)), (3,10)), ( (4,5), ( (6,7), (8,9)))), (12, (16,20))), (14,15)), ( (19, (17,18)), (21, (22,23)))), (24,25)), (26, (29, (27,28)))))) B4 (30, ( (1,26), ( ( ( ( ( ( ( (13, (2,11)), (3,10)), ( (4,5), ( (6,7), (8,9)))), (12, (16,20))), (14,15)), ( (19, (17,18)), (21, (22,23)))), (24,25)), (29, (27,28))))) B5 (30, (1, ( ( (26, ( ( ( ( ( (13, (2,11)), (3,10)), ( (4,5), ( (6,7), (8,9)))), (12, (16,20))), (14,15)), ( (19, (17,18)), (21, (22,23))))), (24,25)), (29, (27,28))))) B6 (30, (1, ( ( ( (26, ( ( ( ( (13, (2,11)), (3,10)), ( (4,5), ( (6,7), (8,9)))), (12, (16,20))), (14,15))), ( (19, (17,18)), (21, (22,23)))), (24,25)), (29, (27,28))))) B7 (30, (1, ( ( ( ( ( ( ( (13, (2,11)), (3,10)), ( (4,5), ( (6,7), (8,9)))), (12, (16,20))), (14,15)), (26, ( (19, (17,18)), (21, (22,23))))), (24,25)), (29, (27,28))))) B8 (30, (1, ( ( ( ( ( ( ( (13, (2,11)), (3,10)), ( (4,5), ( (6,7), (8,9)))), (12, (16,20))), (14,15)), ( (19, (17,18)), (21, (23, (22,26))))), (24,25)), (29, (27,28))))) B9 (30, (1, ( ( ( ( ( ( ( ( (13, (2,11)), (3,10)), ( (4,5), ( (6,7), (8,9)))), (12, (16,20))), (14,15)), ( (19, (17,18)), (21,23))), (24,25)), (22,26)), (29, (27,28))))) B10 (30, (1, ( (26, ( ( ( ( ( ( (2,11), ( (4,5), ( (6,7), (8,9)))), (13, (3,10))), (12, (16,20))), (14,15)), ( (19, (17,18)), (21, (22,23)))), (24,25))), (29, (27,28))))) B11 (30, (1, ( (26, ( ( ( ( ( (13, ( (2,11), ( (4,5), ( (6,7), (8,9))))), (3,10)), (12, (16,20))), (14,15)), ( (19, (17,18)), (21, (22,23)))), (24,25))), (29, (27,28))))) B12 (30, (1, ( (26, ( ( ( ( ( ( (13, (2,11)), (3,10)), ( ( (4,5), (8,9)), (6,7))), (12, (16,20))), (14,15)), ( (19, (17,18)), (21, (22,23)))), (24,25))), (29, (27,28))))) B13 (30, (1, ( (26, ( ( ( ( ( ( (13, (2,11)), (3,10)), ( ( (4,5), (6,7)), (8,9))), (12, (16,20))), (14,15)), ( (19, (17,18)), (21, (22,23)))), (24,25))), (29, (27,28))))) B14 (30, (1, ( (26, ( ( ( ( ( ( (13, (2,11)), (3,10)), ( (4,5), ( (6,7), (8,9)))), (12, (16,20))), (14,15)), ( (17,19), (18, (21, (22,23))))), (24,25))), (29, (27,28)))))

Appendix G. Estimates of divergence times

Since our results indicated younger ages of most anuran cladogenetic events within the Anura as compared to previous publications, we followed a multiple conservative approach, to avoid any methodological error that could mask our results. This implied (a) using the oldest available geological dating of the origin of Mayotte for calibration of the split between the Comoroan Boophis and Mantidactylus from their Malagasy sister groups (8.7 MYr ago), (b) usage of an extremely old age estimate of separation between salamanders and frogs (370 million years, the age of the first tetrapod fossils) (15), (c) usage of the oldest age estimate of the breakup of Africa and South America (101 million years) (16) to calibrate the vicariance between the fully freshwater aquatic Hymenochirus (Africa) and Pipa (South America) (17).

We followed two separate approaches to estimate the ages of the relevant splits in our phylogeny: (i) non-parametric rate smoothing using the program r8s (written by M. J. Sanderson) including the calculation of confidence intervals; as input we used a phylogram with the topology obtained by ML searches of the complete (mitochondrial and nuclear) dataset but with branch lengths based on the nuclear gene sequences only. (ii) Regression of pairwise divergences between taxa in the rhodopsin gene, using additional vicariant species pairs from the Mediterranean area for calibration, and calculation of prediction confidence intervals (18).

7.1 Non Parametric Rate Smoothing

Maximum Likelihood (ML) heuristic searches were carried out with PAUP* (6) under the substitution model proposed by Modeltest (4), with a random sequence addition sequence (10 replicates). The obtained tree was saved without branch lengths, and loaded again after excluding of all mitochondrial sequences and constraining to ML settings proposed by MODELTEST for the nuclear dataset alone. The obtained phylogram was saved to a treefile including branch lengths, and submitted to non parametric rate smoothing (19) using the program r8s, written by M. J. Sanderson, on a Pentium III computer with Linux 2.2.18 partition. We preferred NPRS over penalized likelihood (PL) (20) following again a conservative approach, since it usually produces more extended confidence intervals. NPRS calculations were carried out with the Powell algorithm, fixing the age of the root at 370 or 250 MYA,

the ages of the splits between the two Boophis and the two Mantidactylus at 8.7 MYA, and the age of the split between Pipa and Hymenochirus at 101 MYA. Confidence intervals were calculated using a cutoff value of 4.0. However, due to the lack of crossover points, confidence intervals could not be calculated for the young dispersal events but only for the older splits in the cladogram.

Root fixed at 250 MYr ago Root fixed at 370 MYr ago Fixed ages used for calibration Root 250 MYr ago 370 MYr ago Pipidae 101 MYr ago 101 MYr ago Boophis (Mayotte-Madagascar) 8.7 MYr ago 8.7 MYr ago Mantidactylus (Mayotte-Madagascar) 8.7 MYr ago 8.7 MYr ago

Age estimate Hyperolius-Heterixalus 22.05 MYr ago 26.55 Heterixalus-Tachycnemis 11.28 MYr ago 13.57 Hoplobatrachus occipitalis - H. crassus 8.94 MYr ago 10.29 Rana galamensis - R. temporalis 23.03 MYr ago 26.44 Polypedates - Chiromantis 28.37 MYr ago 32.70

Age estimate and confidence intervals Nesomantis - Neobatrachia 150.50 (118.07-180.56) MYr ago 194.40 (127.61-273.17) MYr ago Hyloidea-Ranoidea 137.57 (105.63-168.39) MYr ago 176.16 (111.13-255.81) MYr ago Ranidei-Arthroleptoidei 92.71 (66.73-126.75) MYr ago 113.63 (70.88-113.68) MYr ago Basal splits within Ranidei 62.79 (41.78-96.99) MYr ago 73.34 (44.96-149.52) MYr ago

7.2 Regression analysis of pairwise Rhodopsin divergences

The calibration of the phylogram submitted to NPRS (see above) was based on three different splits that could be geologically dated (two colonizations of the Comoros, and the vicariance split between the African and South American pipids). To confirm these calibrations with independent data, we sequenced homologous rhodopsin fragments in three species pairs of the Mediterranean region which are likely to have been separated by vicariance at the end of the Messininian salinity crisis of the Mediterranean Sea, 5.2-5.3 MYr ago (21, 22): Rana cretensis (Crete) vs. Rana cerigensis (Rhodos), Pelobates cultripes (Spain) vs. Pelobates varaldii (Morocco), Alytes dickhillenii (Spain) vs. Alytes maurus (Morocco). The two Rana species belong to the European green frogs (or water frogs), which have been successfully used to calibrate a protein clock based on geologically dated Mediterranean sea barriers (21), making it highly probable that the assumed age reflects their biogeographic history. The two Pelobates and Alytes species, respectively, are sister species within their genera (22), and their separation is likelily to have occurred at the opening of the strait of Gibraltar.

The 1-2 substitutions which were consistently found between the species of these pairs correspond well to the 5 substitutions between the Comoroan and Malagasy Mantidactylus species, indicating a rhodopsin substitution rate of 0.1-0.3 substitutions (0.03-0.1%) per lineage per MYr. This agrees with the rate calculated using the ancient split between Xenopus and Pipa (0.2 substitutions per lineage per MYr, corresponding to 0.06%). It also agrees with the placement of the diapsid/synapsid split at 310 MYr ago (23) as exemplified by the Homo/Gallus divergence of 62 substitutions (0.1 substitutions or 0.03% per lineage per MYr).

Since the amount of substitutions between the two Rana (three substitutions) and Hoplobatrachus (three substitutions), as well as between the hyperoliids Heterixalus, Tachycnemis and Hyperolius (6-9 substitutions) were in a similar order of magnitude as in the Mediterranean and Comoran taxa used for calibration, it is highly improbable (and could clearly be excluded by 95% confidence intervals) that these divergences are due to ancient (Mesozoic) vicariance. Such vicariance, for the three hyperoliids, would imply a dramatic three to ten-fold decrease of substitution rate (down to 0.01% per lineage per MYr) for which there is no indication in the branch lengths of the phylograms (see section 5).

Calibration Pairwise divergences Age

(# substitutions) (MYr ago)

Pelobates varaldi - P. cultripes 2 5.3 Alytes maurus - A. dickhillenii 1 5.3 Rana cretensis - R. cerigensis 1 5.3 Mantidactylus wittei - M. sp. (Mayotte) 5 8.7 Xenopus - Pipa 37 101

Age estimate Pairwise divergences Age_mean Age_min Age_max (# substitutions) (MYr ago) (MYr ago) (MYr ago) Chiromantis - Polypedates 16 43.1471 36.3026 50.7792 Rana temporalis - R. galamensis 3 7.9301 3.9599 12.6879 Hoplobatrachus occidentalis - H. crassus 3 7.9301 3.9599 12.6879 Tachycnemis - Heterixalus 6 16.0571 11.4236 21.4782 Heterixalus - Hyperolius 9 24.1841 18.8873 30.2685 Nesomantis - Neobatrachia Min 41 110.8721 98.5001 124.0317 Nesomantis - Neobatrachia Max 49 132.5441 118.4033 147.4725 Neobatrachia Min 33 89.2001 78.5969 100.5909 Neobatrachia Max 51 137.9621 123.3791 153.3327 Ranoidea Min 29 78.3641 68.6453 88.8705 Ranoidea Max 42 113.5811 100.988 126.9618 Ranidei Max 33 89.2001 78.5969 100.5909 Mantellidae-Rhacophoridae Min 10 26.8931 21.3752 33.1986 Mantellidae-Rhacophoridae Max 16 43.1471 36.3026 50.7792

Appendix H. A survey of the terrestrial and freshwater vertebrate families of Madagascar, their biogeographic relationships and fossil ages

Vertebrate group Name of Malagasy taxon non-Malagasy sister taxon Distribution of non- Area cladogram First fossil record of family *** Malagasy sister taxon agreement? (or higher inclusive taxon) Osteichthyes: Cichlidae Paretroplus Etroplus (24) India-Sri Lanka (24) Yes (24, 54) Eocene (25) Osteichthyes: Aplocheiloidea Pachypanchax Oryzias (26) India-Sri Lanka (26) Yes (26) Cyprinodontiformes: Eocene (27, 28)

Amphibia: Hyperoliidae Heterixalus Hyperolius - Afrixalus (3) Africa (3) No (3) Ranoidea: Cretaceous (Cenomanian, 90-97 MYr ago) (29)** Amphibia: Ranoidei Mantellidae Rhacophoridae? (1) India-Asia? (1) Uncertain Ranoidea: Cretaceous (possibly yes) (Cenomanian, 90-97 MYr ago) (29)** Amphibia: Microhylidae Microhylidae: Cophylinae, Microhylidae gen. Unknown Unknown Eocene (29) Dyscophinae, Scaphiophryninae Squamata: Typhlopidae Typhlops spp. Unknown Unknown Unknown Miocene (15) Squamata: Boidae Sanzinia, Acrantophis Boinae gen. (30) South America (30) No (30) Paleocene (31) Squamata: Colubridae Mimophis spp. Psammophiinae* Africa* No* Oligocene (15) Squamata: Chamaeleonidae Calumma, Furcifer Chamaeleo (32, 33) Africa (32, 33) No (32, 33) Paleocene (15) Squamata: Scincidae Mabuya spp. Mabuya spp. (34) Africa (34) No (34) possibly Cretaceous/Jurassic? (15) Squamata: Scincidae Scincinae gen. Scincinae gen.* Africa* No* possibly Cretaceous/Jurassic? (15) Squamata: Gekkonidae Lygodactylus spp. Lygodactylus spp. (35)* Africa (35)* No (35)* Eocene (36) Squamata: Gekkonidae Phelsuma spp. Rhoptropella ocellata (35)* Africa (35),* No (35)* Eocene (36) Squamata: Gekkonidae Blaesodactylus spp. Homopholis spp. (35) Africa (35) No (35) Eocene (36) Squamata: Gerrhosauridae Zonosaurus spp. Gerrhosauridae gen. (37, 38) Africa (37, 38) No (37, 38) Cordylidae s. l. : Eocene (15), maybe Latest Cretaceous? (39) Squamata: Opluridae Opluridae spp. Tropiduridae? South America (40) No (40) Iguanidae s. l.: upper Cretaceous (15, 53) (Iguanidae s.l.) (40) Crocodylia: Crocodylidae Crocodylus niloticus Crocodylus niloticus* Africa* No* Upper Cretaceous (15) Testudines: Podocnemidae Erymnochelys Podocnemis spp. (41, 42) South America (41, 42) No (41, 42) Eocene (41) madagascariensis Testudines: Testudinidae Geochelone spp., Kinixys Geochelone spp. (43) Africa (43) No (43) Cretaceous (Neocomian, 127-144 MYr ago) (44) Mammalia: Rodentia Nesomyinae gen. Muridae gen. (45, 46) Africa (45, 46) No (45, 46) Paleocene (47) Mammalia: Lemuriformes Lemuroidea gen. Lorisidae gen. (48, 49) Africa and Asia (48, 49) No (48, 49) Oligocene (50) Mammalia: Carnivora Herpestidae gen. Herpestidae gen. (47) largely Africa (47) Probably no Paleocene (47) Mammalia: Afrosoricida Tenrecidae gen. Potamogale spp. (51) Africa (51, 52) No (51, 52) Miocene (47)

* According to own observations and unpublished data of A. Schmitz (Bonn)

** Information of ranid fossils of the African Cenomanian have been published (29) but not adequately described so far; in the absence of confirmation, and in the light o recently proposed new classificatons (52), we doubt on the reliability of this familial assignation and refer the findings to the Ranoidea in a preliminary way. *** Seychellean taxa not considered

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