- Email: [email protected]
Phylogenetic position, origin and biogeography of Palearctic and Socotran blind-snakes (Serpentes: Typhlopidae)
Molecular Phylogenetics and Evolution 68 (2013) 35–41
Contents lists available at SciVerse ScienceDirect
Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev
Short Communication
Phylogenetic position, origin and biogeography of Palearctic and Socotran blind-snakes (Serpentes: Typhlopidae) P. Kornilios a,⇑, S. Giokas a, P. Lymberakis b, R. Sindaco c a
Section of Animal Biology, Department of Biology, School of Natural Sciences, University of Patras, GR-26500 Patras, Greece Natural History Museum of Crete, University of Crete, Knossou Ave., 71409 Irakleio, Crete, Greece c c/o Museo Civico di Storia Naturale, via San Francesco di Sales 88, 10022 Carmagnola, Torino, Italy b
a r t i c l e
i n f o
Article history: Received 23 October 2012 Revised 13 February 2013 Accepted 11 March 2013 Available online 21 March 2013 Keywords: Biogeography Letheobia Molecular clock Scolecophidia Squamata
a b s t r a c t The majority of the family Typhlopidae occurs in the Neotropic, Australasian, Indo-Malayan and Afrotropic ecoregions. They show a restricted distribution in the western Palearctic, where they include few native species, i.e. Rhinotyphlops simoni, R. episcopus and Typhlops vermicularis. A unique species among typhlopids is T. socotranus, found in Socotra, one of the most endemic-rich archipelagoes. In this study we determine the phylogenetic position of the above mentioned species and discuss their systematics, origin and biogeography. For this purpose we use three protein-coding nuclear markers (AMEL-amelogenin, BDNF-brain-derived neurotrophic factor and NT3-neurotrophin 3) to construct a time-calibrated phylogeny of the family Typhlopidae. Our results show that T. socotranus is a sister-species to T. vermicularis, while R. simoni and R. episcopus are sister-species to each other and are found within the African clade of the family, although they are geographically distributed in west Asia. Additionally we discuss several hypotheses on their origin, as well as the occurence of typhlopids in Eurasia. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Reptiles and amphibians are regarded as model organisms for biogeographical studies due to their presumed limited dispersal capacities and temperature dependence. Several recent studies of molecular phylogeny have shed new light on the biogeography of herptiles (for a review see Datta-Roy and Karanth, 2009), with plate tectonics, terrestrial dispersal and transoceanic dispersal generally assumed to be the dominant forces shaping largescale patterns of their biogeography (van der Meijden et al., 2007 and references therein). Typhlopidae is the most speciose scolecophidian family, with approximately 250 known species found on all continents except Antarctica (Fig. 1), and presents a typical Gondwanan distribution. The vast majority of its species occurs in the Neotropic, Australasian, Indo-Malayan and Afrotropic ecoregions, whereas Typhlopids show a restricted and isolated distribution in the western Palearctic (Fig. 1) where only a few native species are found. Specifically, the greater part of the western Palearctic distribution of typhlopids is covered by Typhlops vermicularis Merrem, 1820, which most probably represents a species-group rather than a single species: molecular data support the existence of several evolutionary significant units within this taxon, with at least one of
⇑ Corresponding author. E-mail address: [email protected] (P. Kornilios). 1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2013.03.009
them being highly genetically differentiated at a higher-than-species level (‘‘clade A’’ in Kornilios et al., 2012). Additionally, within this range, two species belonging to the genus Rhinotyphlops, i.e. R. simoni (Boettger, 1879) and R. episcopus Franzen & Wallach, 2002, are also found. These exhibit a very rescrticted distribution (Fig. 1) and, although found in west Asia, they belong in an genus distributed in sub-Saharan Africa. Finally, the list of western Palearctic taxa is completed with the alien Ramphotyphlops braminus Daudin 1803, a parthenogenetic species introduced around the world through the nursery trade from Sri Lanka or south India (Wallach, 2008), and Typhlops wilsoni Wall, 1908, known only from the currently untraceable holotype from southwestern Iran (Das and Chaturvedi, 1998) and whose validity as a species is doubtful. A unique species among typhlopids, in terms of its geographic distribution, is Typhlops socotranus Boulenger, 1889. It is found in Socotra, an island of continental origin situated in the north-west Indian Ocean, near the Gulf of Aden (Fig. 1) and considered one of the most remote and most endemic-rich archipelagoes in the world. Very recently, Vidal et al. (2010) reconstructed the first and only large scale phylogeny of Typhlopidae, using nuclear protein-coding markers and including approximately 35% of currently recognized species. Typhlopids were grouped in four geographically-defined clades with unresolved relationships. These correspond to Africa, south America, Madagascar and Eurasia/Australasia. Although the first three regions, east Asia and Australia were well represented, important west Asian taxa were not included. In this context, the
36
P. Kornilios et al. / Molecular Phylogenetics and Evolution 68 (2013) 35–41
Fig. 1. Map showing the geographic distribution of the family Typhlopidae with emphasis on the distribution of the target-species (Zug et al., 2001). The Palearctic ecoregion is presented with a dashed line.
aim of the present study is to determine the phylogenetic position of the above mentioned typhlopid species which have not been previously analyzed and, subsequently, draw conclusions on their systematics, origin and biogeography. Specific questions we aim to address are: (a) What is the phylogenetic position of T. socotranus, does it have Asian or African affinities and what is the underlying biogeographic scenario that could explain its occurrence on Socotra? (b) Is the high mtDNA divergence of T. vermicularis’ ‘‘Clade A’’ also supported by the analysis of nuclear markers in a completely different framework (multi-species phylogeny)? (c) Are R. simoni and R. episcopus grouped with the other sub-Saharan Rhinotyphlops or is this another case of paraphyly and morphological convergence within Typhlopidae? In the former case, what can we hypothesize about their occurrence in Asia and what is their interspecific relationship? Finally, if possible, we aim to produce new information on the biogeography of the entire Typhlopidae family and specifically their occurence in Asia. 2. Material and methods 2.1. Taxon sampling, DNA extraction, amplification and sequencing A total of 80 specimens were analyzed in the present study. These belong to 76 typhlopid species, and Gerrhopilus mirus (Gerrhopilidae) and Xenotyphlops grandidieri (Xenotyphlopidae), which were used as outgroups. Specimen data (codes, sampling localities and GenBank Accession Numbers) are given in Appendix A. Total genomic DNA was extracted with the NucleoSpin Tissue kit (MACHEREY – NAGEL). Partial sequences of three protein-coding nuclear markers were amplified using primers from Vidal et al. (2010): amelogenin (AMEL), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3). PCR products were purified with the NucleoSpin Extract II DNA purification kit (MACHEREY – NAGEL). Sequencing was conducted on an ABI PRISM 3100 capillary sequencer (CEMIA, Larissa, Greece) using the primers of the amplification procedure. Sequences were aligned in ClustalX v.2.0.12 (Larkin et al., 2007) with default parameters. Phylogenies were built using Bayesian Inference (BI) and Maximum Likelihood (ML) with three separate models of evolution for the three codon positions (HKY+G for first and second position,
Hasegawa et al., 1985; K81+G for third position, Kimura, 1981). The selection of the most suitable model of DNA substitution was done with jModeltest v.0.1.1 (Posada, 2008), under the Bayesian Information Criterion. Bayesian analysis was performed in MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003), with eight incrementally heated Markov chains for 3 106 generations, in four independent runs. Substitution-model parameters were unlinked across partitions, and each subset was allowed to have its own rate (prset = variable). After stationarity had been reached, the first 10% of the trees (burnin in Bayesian terms) were discarded, and a majority-rule consensus tree was generated from the post-burnin trees. Partitioned ML analysis was carried out with RAxML 7.2.7 (Stamatakis, 2006), with each partition having its own GTRGAMMA model (Stamatakis, 2006). Nodal support of the tree was tested via 1000 bootstrap replicates. 2.2. Estimation of divergence times To estimate divergence times, we used BEAST v1.6.2 (Drummond and Rambaut, 2007) under an uncorrelated lognormal relaxed molecular clock and a Yule prior on rates of cladogenesis. Four runs were conducted with a chain length of 10 106 iterations and a burn-in of 2 106 iterations. The four runs were analyzed in TRACER (Rambaut and Drummond, 2007) to check for convergence of the chains and combined in LogCombiner from the BEAST package. TreeAnnotator, also from the BEAST package, was used for the production of the chronogram. Vidal et al. (2010) used a combination of calibration points for their divergence-times estimation. These included fossil records, geological events and secondary calibration points. In our analysis, we used several secondary calibration points, as resulted from Vidal et al. (2010), shown in Fig. 2. 3. Results and discussion The phylogenetic trees (BI and ML) and estimated divergence dates recovered here (Fig. 2; Supplementary material Fig. S1) were identical to the ones of Vidal et al. (2010) (their Fig. 1). Regarding the molecular dating, our calibration points corresponded to strongly supported nodes situated within the groups of Madagas-
P. Kornilios et al. / Molecular Phylogenetics and Evolution 68 (2013) 35–41
37
Fig. 2. (A) The phylogenetic tree produced in the present study (BI tree shown) where all values of statistical support for the strongly supported nodes are presented (BI posterior probabilities above branches, ML bootstrap values below branches). Outgroups, Gerrhopilus mirus and Xenotyphlops grandidieri are not shown. (B) White circles indicate selected nodes whose age in My is shown in this inset (mean estimated times and 95% intervals). Black circles indicate calibration points for the molecular clock analysis. Mean ages and intervals for all nodes are shown in the Supplementary material in Fig. S1.
car, South America, West Indies and Australia (i.e. we avoided calibrating nodes within the groups of interest, Eurasia and Africa). The use of secondary calibrations has received deserved criticism when used as errorless point calibrations (see Graur and Martin, 2004). However, Bayesian age estimation allows for the explicit incorporation of this error by permitting age calibration constraints in the form of statistical distributions (Kuriyama et al., 2011). In this context, we have used the statistical distributions (mean, standard deviation) inferred by the primary analysis of Vi-
dal et al. (2010), rather than simple point calibrations, in order to account for this error. Finally, there are several inconsistencies between the phylogenetic groupings (Vidal et al., 2010; present study), based on the nuclear marker analysis, and the current taxonomy of the Typhlopidae. It seems that, in many cases, the morphological characters used for the description of typhlopid genera were not suitable and are probably the result of convergent phenotypic evolution. However, since the present study does not aim to make
38
P. Kornilios et al. / Molecular Phylogenetics and Evolution 68 (2013) 35–41
any taxonomic re-evaluations, we use current taxonomy and nomenclature. 3.1. Typhlops vermicularis and ‘‘clade A’’ In a recent study, mtDNA markers distinguished several T. vermicularis’ evolutionary significant units (ESUs), one of which, named ‘‘clade A’’, was largely differentiated (Kornilios et al., 2012). The nuclear marker of the prolactin receptor (PRLR) also supported its genetic differentiation, but showed no variation among the remaining ESUs (Kornilios et al., 2012). In the same study, a vicariant event was inferred, having occurred in the Middle East and resulting in the split of ‘‘clade A’’ from the other T. vermicularis. This split was dated approximately 9.8 Mya (95% intervals: 7.7–14.2 Mya), in the Tortonian, when a major aridification/cooling step of the Cenozoic climatic history occurred. This large-scale aridification is probably explained by the Tibetan plateau/Himalayan uplift, as studies point to 10–8 Mya as the age when high topographies were attained in south Asia (An et al., 2001). The analysis of new nuclear markers in a different framework in the current study confirms the results from the mtDNA markers. This lineage (Fig. 2) exhibits high levels of genetic diversity from the other T. vermicularis, while the time of its phylogenetic split is very old compared to other morphologically distinct and recognized typhlopid species (Fig. 2). Additionally, the age of this split from the nDNA analysis, estimated some 9.2 Mya (3.8–16.0 Mya), largely agrees to that of the mtDNA, although their estimation was based on different independent markers and resulted from very different calibration strategies (use of a single calibration point for the mtDNA, based on the palaeogeography of eastern Mediterranean). 3.2. Rhinotyphlops All Rhinotyphlops species are included in the African clade (Vidal et al., 2010; current study). Rhinotyphlops (and the morphologically similar Letheobia) is distributed in sub-Saharan Africa (Broadley and Wallach, 2007). However, two species are distributed in west Asia, i.e. R. episcopus and R. simoni, occurring in Turkey and the Middle East, respectively (Fig. 1). Although analysis of morphological characters has led to the inclusion of several former Rhinotyphlops species to the genus Letheobia, the two mentioned species, along with others, have not been investigated yet. Our results show that they are in fact found within the African clade and related to African Rhinotyphlops, forming a monophyletic group with R. newtoni and R. feae (bootsrap values = 100, pp = 1.0) (Fig. 2). It is worth noting that R. newtoni and R. feae, which occur on the volcanic island of São Tomé at the gulf of Guinea (Fig. 1), are also morphologically similar to the west Asian Rhinotyphlops (Roux-Estève, 1974). The estimated time of divergence between R. episcopus/R. simoni and R. newtoni/R. feae is in the Early Miocene, approximately 19.6 Mya (11.7–29.1 Mya). This time corresponds to the time of occurrence of their most recent common ancestor (MRCA). Unfortunately, no representatives of this particular subgroup from continental Africa are included in our analysis, and therefore two conclusions may be drawn: the estimated time of divergence corresponds to (a) the colonization of São Tomé by Rhinotyphlops, or (b) the split between sub-Saharan and Asian Rhinotyphlops. In the first scenario, the colonization of São Tomé by Rhinotyphlops occurred earlier than the isolation of Asian Rhinotyphlops from sub-Saharan ones (an event that cannot be dated). If this scenario is true, then Rhinotyphlops reached São Tomé through overseas dispersal at the very early stages of the island’s formation, since the oldest dated volcanic rocks are 15.7 My old (Deruelle et al., 1991). In the second scenario, it is the colonization
of São Tomé that cannot be dated, occurring some time later, but the isolation of R. simoni and R. episcopus from the African Rhinotyphlops occurred some 19.6 Mya (11.7–29.1 Mya), as a result of a possible vicarianistic event of geologic or climatic origin. Additionally, R. simoni and R. episcopus show a sister-species relationship to each other. R. simoni’s geographic distribution is restricted in Israel, Gaza strip, Golan Heights and western Jordan, while R. episcopus has a restricted distribution in southeastern Turkey (Fig. 1). There is a distributional gap of several hundreds of kilometers between them, while the distribution of R. simoni coincides to great extent with the distribution of T. vermicularis’ ‘‘clade A’’. Thus, the phylogenetic split of R. simoni/R. episcopus and the basal diversification within T. vermicularis seem to have occurred in the same region. Most importantly, they seem to have taken place during the same period, and specifically at 10.0 Mya (4.5– 16.8 Mya) and 9.2 Mya (3.8–16.0 Mya), respectively, implying the act of a common vicarianistic agent, i.e. the major aridification events that happened in the Tortonian (see above for T. vermicularis and ‘‘clade A’’). 3.3. Typhlops socotranus The Socotran archipelago rests on a shelf platform at the triple junction of the Gondwanan plates of India, Arabia and Africa. Tectonic activity in the Red Sea/Gulf of Aden region during the Oligocene marked the onset of the rifting between Arabia and Somalia continental blocks about 30–17.6 Mya (syn-rift) followed by a continental break-up and oceanic spreading at 17.6 Mya (post-rift) that increased the distance between the Socotra Archipelago and Arabian mainland (Autin et al., 2010). The complex geological history of the Socotran archipelago, together with its diverse topology and climate, are primary responsible for its high levels of endemism. Its herpetofauna includes 28 native and two introduced terrestrial species (Razzetti et al., 2011) but few of them have been investigated using appropriate dating methods. Although considered part of the Afrotropic ecoregion, most of its herpetofauna has Palearctic affinities. So far, it seems that Hemidactylus geckos, Trachylepis skinks and Mesalina lizards have colonized Socotra island from Arabia after its complete isolation (Gómez-Díaz et al., 2012; Sindaco et al., 2012; Joger and Mayer, 2002), while the snakes Hemerophis and Ditypophis and Chamaeleo chameleons were probably present on Socotra when it separated from mainland Arabia (Macey et al., 2008; Nagy et al., 2003). Our analyses show that T. socotranus is related to T. vermicularis, as they form a monophyletic group (bootstrap values = 100, pp = 1.0) (Fig. 2). This result was not anticipated since there is a distributional gap of >1500 km between these two species (>2500 between T. socotranus and T. vermicularis ‘‘clade A’’) (Fig. 1). On the other hand, the sea-barrier between Socotra and the Horn of Africa is only 250 km. Nevertheless, T. socotranus, along with T. vermicularis, lie within the Eurasia/Australasia major clade and not the African one. The time of divergence between T. socotranus and T. vermicularis is estimated to be 29.6 Mya (18.3–42.1 Mya), i.e. the age of their MRCA. This leads to two basic hypotheses for the underlying diversification event: (a) T. socotranus ancestor was isolated on Socotra at the very early stage of the island’s geological isolation (rifting between Arabia and Somalia began some 30 Mya) or (b) the T. vermicularis/T. socotranus ancestor had a much wider distribution in the past covering the Arabian peninsula and, a subsequent extinction event occurring approximately 29.6 Mya, acted as a vicariant agent and isolated populations in the north (T. vermicularis ancestor) and the south (T. socotranus ancestor). Palaeogeographical reconstructions do not seem to support the latter hypothesis (e.g. Popov et al., 2004), while the global climatic-cooling event
39
P. Kornilios et al. / Molecular Phylogenetics and Evolution 68 (2013) 35–41
occurring at the Eocene–Oligocene transition is a possible vicariant agent, as it is responsible for the mass extinctions of this era (Xiao et al., 2010 and references therein). Additionally, in this scenario, Socotra was colonized some time later, but the age of this colonization via overseas dispersal, cannot be definitively inferred. In any case, an extinction event has surely taken place at some point, since there are no typhlopids presently occurring in the Arabian Peninsula (Fig. 1).
3.4. Typhlopid dispersal to Eurasia A diversification event seems to have occurred within the Eurasia/Australasia clade some 61.1 Mya (51.3–70.7 Mya) splitting the western species (T. vermicularis and T. socotranus) from the Indo-Malayan and Australasian ones. According to Vidal et al. (2010), the Eurasian/Australasian group probably originated by a northward dispersal from Gondwana, either out of Africa through Europe and Asia (Laurasia) or out of India. The latter refers to a dispersal event from northeast Africa/Arabia to the drifting subcontinent of India (through land connections or a volcanic island arc) and finally to Asia after the collision of the Indian continent with Asia (see Chatterjee and Scotese, 2010). Both scenarios involve a single dispersal (colonization) event to Eurasia and a subsequent expansion westwards (even reaching Europe) and eastwards (reaching Australia), while the diversification between west and east Asian typhlopids can be explained by a vicarianistic event, occurring some 60 Mya. This event ‘‘broke-up’’ a continuous distribution into a western and an eastern part creating a distributional gap, that remains until today (Fig. 1). There are two problems concerning the hypotheses of a single dispersal (either from Africa or ‘‘out of India’’): (a) there is no known geologic or climatic event occurring at that time (Middle Paleocene) to have acted as a vicariant agent, and (b) especially for the ‘‘out of India’’ scenario, the diversification in east and west groups would have happened within India much earlier than the actual hard collision with Asia (35 Mya) (Ali and Aitchison, 2008). Although we do not reject these two hypotheses, we propose a third one that better fits our results. This involves two dispersal events from the same source, i.e. from north-east Africa/Arabia: one northwards-westwards which led to the west Asian typhlopids and a second one eastwards to India and later to south-east Asia, Working code
Species
Tv7 Rs1
T. vermicularis ‘‘Clade A’’ R. simoni
Re1
R. episcopus
Re2
R. episcopus
Ts1
T. socotranus
Ts2
T. socotranus T. T. T. T. T.
granti hypomethes platycephalus richardi naugus
Museum code
Locality (country)
NHMC 80.3.21.7 NHMC 80.3.21.8 MCCIR1619(1) MCCIR1619(2) MCCIR1432 MCCIR1493
leading to the east typhlopid group (‘‘out of India’’). This scenario explains (a) the monophyly (common ancestry) of Eurasian/Australasian Typhlopidae, (b) the distributional gap between them, since it involves two independent geographic expansions in the west and the east, respectively, and (c) it fits the estimated ages better. This hypothesis is also backed-up by the fact that the eastern part of the west-Asian typhlopids’ distribution (Fig. 1) is very recent (Middle or Late Pleistocene) (Kornilios et al., 2012), while the most morphologically similar species to T. vermicularis is the northwest African T. etheridgei (Wallach, 2002). Conclusively, blindsnakes (Typhlopoidea) have managed to disperse into Asia from Africa at least four times. A very early dispersal is described by the ‘‘out of India hypothesis’’ also known as the ‘‘Biotic ferry model’’. According to this, the rafting Indian plate carried ancient Gondwanan forms to Asia, now recognized as a separate family, the Gerrhopilidae (Vidal et al., 2010). During its ‘‘journey’’, a second dispersal occurred from northeast Africa/ Arabia to the rafting Indian subcontinent and subsequently to Asia after its collision. This led to the east Asia/Austalasia typhlopid radiation. A third dispersal from the same region to west Eurasia gave rise to the west Asia radiation (T. vermicularis species complex and T. socotranus). Finally, a forth dispersal from Africa to west Asia resulted in the asian Rhinotyphlops species.
Acknowledgments R.S. acknowledges for their help during field work Cristina Grieco and Elisa Riservato (Anatolia and Socotra), Fabio Pupin, Edoardo Razzetti and Mauro Fasola (Socotra).
Appendix A. Sample working codes, species names, museum numbers (NHMC: Natural History Museum of Crete; MCC: Museo Civico di Storia Naturale di Carmagnola, Italy) and sampling locations (locality/country) of the specimens analyzed in the current study. GenBank accession numbers of sequence data for the DNA segments are also shown (AMEL: amelogenin, BDNF: brain-derived neurotrophic factor, NT3: neurotrophin 3). GenBank accession number AMEL
BDNF
NT3
Zai Park (Jordan) 32.150037, 35.716553
KC848442
KC848451
KC848454
4 km S of Al Mazar, al Janubi (Jordan) 31.052934, 35.612183 S ß anliurfa prov., Halfeti env. (Turkey) 37.246181, 37.868156 S ß anliurfa prov., Halfeti env. (Turkey) 37.246181, 37.868156 Sokotra Isl., between Eerk and Jelhiiyo (Yemen) 12.567968, 54.162769 Sokotra Isl., Mala Plateau (Yemen) 12.655078, 53.491058
KC848447
KC848448
KC848459
KC848445
KC848449
KC848457
KC848446
KC848450
KC848458
KC848443
KC848452
KC848455
KC848444
KC848453
KC848456
GU902350 GU902351 GU902357 GU902358 GU902355
GU902430 GU902431 GU902437 GU902438 GU902435
GU902600 GU902601 GU902607 GU902608 GU902605
(continued on next page)
40
P. Kornilios et al. / Molecular Phylogenetics and Evolution 68 (2013) 35–41
(continued)
Working code
Species
T. catapontus T. dominicanus T. geotomus T. monastus T. caymanensis T. contorhinus T. anousius T. notorachius T. arator T. anchaurus T. rostellatus T. syntherus T. sulcatus T. capitulatus T. jamaicensis T. sylleptor T. agoralionis T. lumbricalis T. eperopeus T. schwartzi T. reticulatus T. brongersmianus T. angolensis T. obtusus T. elegans T. bibronii T. fornasinii T. lineolatus T. congestus T. punctatus R. schlegelii R. mucruso R. newtoni R. feae R. lalandei R. unitaeniatus T. luzonensis R. albiceps Ramphotyphlops sp. R. braminus T. pammeces R. lineatus R. acuticaudus Acutotyphlops sp. A. subocularis A. kunuaensis R. polygrammicus R. longissimus R. bituberculatus R. unguirostris R. kimberleyensis R. ganei R. ligatus R. bicolor R. splendidus R. pinguis R. hamatus R. australis R. pilbarensis R. waitii R. diversus
Museum code
Locality (country)
GenBank accession number AMEL
BDNF
NT3
GU902346 GU902348 GU902349 GU902354 GU902347 GU902366 GU902365 GU902356 GU902344 GU902343 GU902359 GU902363 GU902361 GU902345 GU902352 GU902362 GU902342 GU902353 GU902364 GU902360 GU902319 GU902313 GU902312 GU902375 GU902314 GU902370 GU902367 GU902371 GU902368 GU902318 GU902369 GU902310 GU902311 GU902308 GU902309 GU902372 GU902316 GU902305 GU902340 GU902306 GU902378 GU902307 GU902304 GU902379 GU902338 GU902339 GU902341 GU902330 GU902325 GU902329 GU902328 GU902334 GU902327 GU902332 GU902337 GU902336 GU902323 GU902331 GU902322 GU902324 GU902333
GU902426 GU902428 GU902429 GU902434 GU902427 GU902446 GU902445 GU902436 GU902424 GU902423 GU902439 GU902443 GU902441 GU902425 GU902432 GU902442 GU902422 GU902433 GU902444 GU902440 GU902396 GU902390 GU902389 – GU902314 GU902450 GU902617 GU902451 GU902448 GU902395 GU902449 GU902387 GU902388 GU902385 GU902386 GU902452 GU902393 GU902382 GU902420 GU902383 GU902458 GU902384 GU902381 GU902459 GU902418 GU902419 GU902421 GU902408 GU902403 GU902407 GU902406 GU902412 GU902405 GU902410 GU902416 GU902415 GU902401 GU902409 GU902400 GU902402 GU902411
GU902596 GU902598 GU902599 GU902604 GU902597 GU902616 GU902615 GU902606 GU902594 GU902593 GU902609 GU902613 GU902611 GU902595 GU902602 GU902612 GU902592 GU902603 GU902614 GU902610 GU902568 GU902563 GU902562 GU902625 GU902564 GU902620 GU902447 GU902621 GU902618 GU902567 GU902619 GU902560 GU902561 GU902558 GU902559 – – GU902555 – GU902556 GU902628 GU902557 GU902554 GU902629 GU902589 GU902590 GU902591 GU902579 GU902574 GU902578 GU902577 GU902583 GU902576 GU902581 GU902587 GU902586 GU902572 GU902580 GU902571 GU902573 GU902582
41
P. Kornilios et al. / Molecular Phylogenetics and Evolution 68 (2013) 35–41 (continued)
Working code
Species
Museum code
Locality (country)
R. howi R. guentheri T. arenarius T. andasibensis X. grandidieri G. mirus
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2013.03. 009. References Ali, J.R., Aitchison, J.C., 2008. Gondwana to Asia: plate tectonics, paleogeography and the biological connectivity of the Indian sub-continent from the Middle Jurassic through latest Eocene (166–35 Ma). Earth Sci. Rev. 88, 145–166. An, Z., Kutzbach, J.E., Prell, W.L., Porter, S.C., 2001. Evolution of Asian monsoons and phased uplift of the Himalaya-Tibetan Plateau since late Miocene times. Nature 411, 62–66. Autin, J., Leroy, S., Beslier, M.-O., d’Acremont, E., Razin, P., Ribodetti, A., Bellahsen, N., Robin, C., Al Toubi, K., 2010. Continental break-up history of a deep magmapoor margin based on seismic reflection data (northeastern Gulf of Aden margin, offshore Oman). Geophys. J. Int. 180, 501–519. Broadley, D.G., Wallach, V., 2007. A review of East and Central African species of Letheobia Cope, revived from the synonymy of Rhinotyphlops Fitzinger, with descriptions of five new species (Serpentes: Typhlopidae). Zootaxa 1515, 31–68. Chatterjee, S., Scotese, C., 2010. The wandering Indian plate and its changing biogeography during the late cretaceous-early tertiary period. In: Bandyopadhyay, S. (Ed.), New Aspects of Mesozoic Biodiversity, Lecture Notes in Earth Sciences. Springer, Berlin/Heidelberg, pp. 105–126. Das, I., Chaturvedi, N., 1998. Catalogue of herpetological types in the collection of the bombay Natural History Society. Hamadryad 23, 150–156. Datta-Roy, A., Karanth, K.P., 2009. The out-of-India hypothesis: what do molecules suggest? J. Biosci. 34, 687–697. Deruelle, B., Moreau, C., Nkoumbou, C., Kambou, R., Lissom, J., Njongfang, E., Ghogomu, R.T., Nono, A., 1991. The Cameroon Line: a review. In: Kampunzu, A.B., Lubala, R.T. (Eds.), Magmatism in Extensional Structural Settings. Springer Verlag, Berlin, Germany, pp. 274–327. Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214. Gómez-Díaz, E., Sindaco, R., Pupin, F., Fasola, M., Carranza, S., 2012. Origin and in situ diversification in Hemidactylus geckos of the Socotra Archipelago. Mol. Ecol. 21, 4074–4092. Graur, D., Martin, W., 2004. Reading the entrails of chickens: molecular timescales of evolution and the illusion of precision. Trends Genet. 20, 80–86. Hasegawa, M., Kishino, K., Yano, T., 1985. Dating the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22, 160–174. Joger, U., Mayer, W., 2002. A new species of Mesalina (Reptilia: Lacertidae) from Abd al-Kuri, Socotra Archipelago, Yemen, and a preliminary molecular phylogeny for the genus Mesalina. Fauna Arab. 19, 497–505. Kimura, M., 1981. Estimation of evolutionary distances between homologous nucleotide sequences. Proc. Natl. Acad. Sci. USA 78, 454–458. Kornilios, P., Ilgaz, Ç., Kumlutas, Y., Lymberakis, P., Moravec, J., Sindaco, R., RastegarPouyani, N., Afroosheh, M., Giokas, S., Fraguedakis-Tsolis, S., Chondropoulos, B., 2012. Neogene climatic oscillations shape the biogeography and evolutionary history of the Eurasian blindsnake. Mol. Phylogenet. Evol. 62, 856–873.
GenBank accession number AMEL
BDNF
NT3
GU902335 GU902326 GU902374 GU902373 GU902377 GU902317
GU902414 GU902404 GU902455 GU902453 GU902457 GU902394
GU902585 GU902575 GU902624 GU902622 GU902627 GU902566
Kuriyama, T., Brandley, M.C., Katayama, A., Mori, A., Honda, M., Hasegawa, M., 2011. A time-calibrated phylogenetic approach to assessing the phylogeography, colonization history and phenotypic evolution of snakes in the Japanese Izu Islands. J. Biogeogr. 38, 259–271. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948. Macey, J.R., Kuehl, J.V., Larson, A., Robinson, M.D., Ugurtas, I.H., Ananjeva, N.B., Rahman, H., Javed, H.I., Osman, R.M., Doummak, A., Papenfuss, T.J., 2008. Socotra Island the forgotten fragment of Gondwana: Unmasking chameleon lizard history with complete mitochondrial genomic data. Mol. Phylogenet. Evol. 49, 1015–1018. Nagy, Z.T., Joger, U., Wink, M., Glaw, F., Vences, M., 2003. Multiple colonization of Madagascar and Socotra by colubrid snakes: evidence from nuclear and mitochondrial gene phylogenies. Proc. R. Soc. Lond. B Biol. Sci. 270, 2613–2621. Popov, S.V., Rögl, F., Rozanov, A.Y., Steiniger, F.F., Shcherba, I.G., Kovac, M., 2004. Lithological-paleogeographic maps of Paratethys: 10 maps Late Eocene to Pliocene. Cour. Forsch.-Inst. Senckenberg 250, 1–46. Posada, D., 2008. JModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25, 1253–1256. Rambaut, A., Drummond, A.J., 2007. Tracer v1.5.
Contents lists available at SciVerse ScienceDirect
Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev
Short Communication
Phylogenetic position, origin and biogeography of Palearctic and Socotran blind-snakes (Serpentes: Typhlopidae) P. Kornilios a,⇑, S. Giokas a, P. Lymberakis b, R. Sindaco c a
Section of Animal Biology, Department of Biology, School of Natural Sciences, University of Patras, GR-26500 Patras, Greece Natural History Museum of Crete, University of Crete, Knossou Ave., 71409 Irakleio, Crete, Greece c c/o Museo Civico di Storia Naturale, via San Francesco di Sales 88, 10022 Carmagnola, Torino, Italy b
a r t i c l e
i n f o
Article history: Received 23 October 2012 Revised 13 February 2013 Accepted 11 March 2013 Available online 21 March 2013 Keywords: Biogeography Letheobia Molecular clock Scolecophidia Squamata
a b s t r a c t The majority of the family Typhlopidae occurs in the Neotropic, Australasian, Indo-Malayan and Afrotropic ecoregions. They show a restricted distribution in the western Palearctic, where they include few native species, i.e. Rhinotyphlops simoni, R. episcopus and Typhlops vermicularis. A unique species among typhlopids is T. socotranus, found in Socotra, one of the most endemic-rich archipelagoes. In this study we determine the phylogenetic position of the above mentioned species and discuss their systematics, origin and biogeography. For this purpose we use three protein-coding nuclear markers (AMEL-amelogenin, BDNF-brain-derived neurotrophic factor and NT3-neurotrophin 3) to construct a time-calibrated phylogeny of the family Typhlopidae. Our results show that T. socotranus is a sister-species to T. vermicularis, while R. simoni and R. episcopus are sister-species to each other and are found within the African clade of the family, although they are geographically distributed in west Asia. Additionally we discuss several hypotheses on their origin, as well as the occurence of typhlopids in Eurasia. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Reptiles and amphibians are regarded as model organisms for biogeographical studies due to their presumed limited dispersal capacities and temperature dependence. Several recent studies of molecular phylogeny have shed new light on the biogeography of herptiles (for a review see Datta-Roy and Karanth, 2009), with plate tectonics, terrestrial dispersal and transoceanic dispersal generally assumed to be the dominant forces shaping largescale patterns of their biogeography (van der Meijden et al., 2007 and references therein). Typhlopidae is the most speciose scolecophidian family, with approximately 250 known species found on all continents except Antarctica (Fig. 1), and presents a typical Gondwanan distribution. The vast majority of its species occurs in the Neotropic, Australasian, Indo-Malayan and Afrotropic ecoregions, whereas Typhlopids show a restricted and isolated distribution in the western Palearctic (Fig. 1) where only a few native species are found. Specifically, the greater part of the western Palearctic distribution of typhlopids is covered by Typhlops vermicularis Merrem, 1820, which most probably represents a species-group rather than a single species: molecular data support the existence of several evolutionary significant units within this taxon, with at least one of
⇑ Corresponding author. E-mail address: [email protected] (P. Kornilios). 1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2013.03.009
them being highly genetically differentiated at a higher-than-species level (‘‘clade A’’ in Kornilios et al., 2012). Additionally, within this range, two species belonging to the genus Rhinotyphlops, i.e. R. simoni (Boettger, 1879) and R. episcopus Franzen & Wallach, 2002, are also found. These exhibit a very rescrticted distribution (Fig. 1) and, although found in west Asia, they belong in an genus distributed in sub-Saharan Africa. Finally, the list of western Palearctic taxa is completed with the alien Ramphotyphlops braminus Daudin 1803, a parthenogenetic species introduced around the world through the nursery trade from Sri Lanka or south India (Wallach, 2008), and Typhlops wilsoni Wall, 1908, known only from the currently untraceable holotype from southwestern Iran (Das and Chaturvedi, 1998) and whose validity as a species is doubtful. A unique species among typhlopids, in terms of its geographic distribution, is Typhlops socotranus Boulenger, 1889. It is found in Socotra, an island of continental origin situated in the north-west Indian Ocean, near the Gulf of Aden (Fig. 1) and considered one of the most remote and most endemic-rich archipelagoes in the world. Very recently, Vidal et al. (2010) reconstructed the first and only large scale phylogeny of Typhlopidae, using nuclear protein-coding markers and including approximately 35% of currently recognized species. Typhlopids were grouped in four geographically-defined clades with unresolved relationships. These correspond to Africa, south America, Madagascar and Eurasia/Australasia. Although the first three regions, east Asia and Australia were well represented, important west Asian taxa were not included. In this context, the
36
P. Kornilios et al. / Molecular Phylogenetics and Evolution 68 (2013) 35–41
Fig. 1. Map showing the geographic distribution of the family Typhlopidae with emphasis on the distribution of the target-species (Zug et al., 2001). The Palearctic ecoregion is presented with a dashed line.
aim of the present study is to determine the phylogenetic position of the above mentioned typhlopid species which have not been previously analyzed and, subsequently, draw conclusions on their systematics, origin and biogeography. Specific questions we aim to address are: (a) What is the phylogenetic position of T. socotranus, does it have Asian or African affinities and what is the underlying biogeographic scenario that could explain its occurrence on Socotra? (b) Is the high mtDNA divergence of T. vermicularis’ ‘‘Clade A’’ also supported by the analysis of nuclear markers in a completely different framework (multi-species phylogeny)? (c) Are R. simoni and R. episcopus grouped with the other sub-Saharan Rhinotyphlops or is this another case of paraphyly and morphological convergence within Typhlopidae? In the former case, what can we hypothesize about their occurrence in Asia and what is their interspecific relationship? Finally, if possible, we aim to produce new information on the biogeography of the entire Typhlopidae family and specifically their occurence in Asia. 2. Material and methods 2.1. Taxon sampling, DNA extraction, amplification and sequencing A total of 80 specimens were analyzed in the present study. These belong to 76 typhlopid species, and Gerrhopilus mirus (Gerrhopilidae) and Xenotyphlops grandidieri (Xenotyphlopidae), which were used as outgroups. Specimen data (codes, sampling localities and GenBank Accession Numbers) are given in Appendix A. Total genomic DNA was extracted with the NucleoSpin Tissue kit (MACHEREY – NAGEL). Partial sequences of three protein-coding nuclear markers were amplified using primers from Vidal et al. (2010): amelogenin (AMEL), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3). PCR products were purified with the NucleoSpin Extract II DNA purification kit (MACHEREY – NAGEL). Sequencing was conducted on an ABI PRISM 3100 capillary sequencer (CEMIA, Larissa, Greece) using the primers of the amplification procedure. Sequences were aligned in ClustalX v.2.0.12 (Larkin et al., 2007) with default parameters. Phylogenies were built using Bayesian Inference (BI) and Maximum Likelihood (ML) with three separate models of evolution for the three codon positions (HKY+G for first and second position,
Hasegawa et al., 1985; K81+G for third position, Kimura, 1981). The selection of the most suitable model of DNA substitution was done with jModeltest v.0.1.1 (Posada, 2008), under the Bayesian Information Criterion. Bayesian analysis was performed in MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003), with eight incrementally heated Markov chains for 3 106 generations, in four independent runs. Substitution-model parameters were unlinked across partitions, and each subset was allowed to have its own rate (prset = variable). After stationarity had been reached, the first 10% of the trees (burnin in Bayesian terms) were discarded, and a majority-rule consensus tree was generated from the post-burnin trees. Partitioned ML analysis was carried out with RAxML 7.2.7 (Stamatakis, 2006), with each partition having its own GTRGAMMA model (Stamatakis, 2006). Nodal support of the tree was tested via 1000 bootstrap replicates. 2.2. Estimation of divergence times To estimate divergence times, we used BEAST v1.6.2 (Drummond and Rambaut, 2007) under an uncorrelated lognormal relaxed molecular clock and a Yule prior on rates of cladogenesis. Four runs were conducted with a chain length of 10 106 iterations and a burn-in of 2 106 iterations. The four runs were analyzed in TRACER (Rambaut and Drummond, 2007) to check for convergence of the chains and combined in LogCombiner from the BEAST package. TreeAnnotator, also from the BEAST package, was used for the production of the chronogram. Vidal et al. (2010) used a combination of calibration points for their divergence-times estimation. These included fossil records, geological events and secondary calibration points. In our analysis, we used several secondary calibration points, as resulted from Vidal et al. (2010), shown in Fig. 2. 3. Results and discussion The phylogenetic trees (BI and ML) and estimated divergence dates recovered here (Fig. 2; Supplementary material Fig. S1) were identical to the ones of Vidal et al. (2010) (their Fig. 1). Regarding the molecular dating, our calibration points corresponded to strongly supported nodes situated within the groups of Madagas-
P. Kornilios et al. / Molecular Phylogenetics and Evolution 68 (2013) 35–41
37
Fig. 2. (A) The phylogenetic tree produced in the present study (BI tree shown) where all values of statistical support for the strongly supported nodes are presented (BI posterior probabilities above branches, ML bootstrap values below branches). Outgroups, Gerrhopilus mirus and Xenotyphlops grandidieri are not shown. (B) White circles indicate selected nodes whose age in My is shown in this inset (mean estimated times and 95% intervals). Black circles indicate calibration points for the molecular clock analysis. Mean ages and intervals for all nodes are shown in the Supplementary material in Fig. S1.
car, South America, West Indies and Australia (i.e. we avoided calibrating nodes within the groups of interest, Eurasia and Africa). The use of secondary calibrations has received deserved criticism when used as errorless point calibrations (see Graur and Martin, 2004). However, Bayesian age estimation allows for the explicit incorporation of this error by permitting age calibration constraints in the form of statistical distributions (Kuriyama et al., 2011). In this context, we have used the statistical distributions (mean, standard deviation) inferred by the primary analysis of Vi-
dal et al. (2010), rather than simple point calibrations, in order to account for this error. Finally, there are several inconsistencies between the phylogenetic groupings (Vidal et al., 2010; present study), based on the nuclear marker analysis, and the current taxonomy of the Typhlopidae. It seems that, in many cases, the morphological characters used for the description of typhlopid genera were not suitable and are probably the result of convergent phenotypic evolution. However, since the present study does not aim to make
38
P. Kornilios et al. / Molecular Phylogenetics and Evolution 68 (2013) 35–41
any taxonomic re-evaluations, we use current taxonomy and nomenclature. 3.1. Typhlops vermicularis and ‘‘clade A’’ In a recent study, mtDNA markers distinguished several T. vermicularis’ evolutionary significant units (ESUs), one of which, named ‘‘clade A’’, was largely differentiated (Kornilios et al., 2012). The nuclear marker of the prolactin receptor (PRLR) also supported its genetic differentiation, but showed no variation among the remaining ESUs (Kornilios et al., 2012). In the same study, a vicariant event was inferred, having occurred in the Middle East and resulting in the split of ‘‘clade A’’ from the other T. vermicularis. This split was dated approximately 9.8 Mya (95% intervals: 7.7–14.2 Mya), in the Tortonian, when a major aridification/cooling step of the Cenozoic climatic history occurred. This large-scale aridification is probably explained by the Tibetan plateau/Himalayan uplift, as studies point to 10–8 Mya as the age when high topographies were attained in south Asia (An et al., 2001). The analysis of new nuclear markers in a different framework in the current study confirms the results from the mtDNA markers. This lineage (Fig. 2) exhibits high levels of genetic diversity from the other T. vermicularis, while the time of its phylogenetic split is very old compared to other morphologically distinct and recognized typhlopid species (Fig. 2). Additionally, the age of this split from the nDNA analysis, estimated some 9.2 Mya (3.8–16.0 Mya), largely agrees to that of the mtDNA, although their estimation was based on different independent markers and resulted from very different calibration strategies (use of a single calibration point for the mtDNA, based on the palaeogeography of eastern Mediterranean). 3.2. Rhinotyphlops All Rhinotyphlops species are included in the African clade (Vidal et al., 2010; current study). Rhinotyphlops (and the morphologically similar Letheobia) is distributed in sub-Saharan Africa (Broadley and Wallach, 2007). However, two species are distributed in west Asia, i.e. R. episcopus and R. simoni, occurring in Turkey and the Middle East, respectively (Fig. 1). Although analysis of morphological characters has led to the inclusion of several former Rhinotyphlops species to the genus Letheobia, the two mentioned species, along with others, have not been investigated yet. Our results show that they are in fact found within the African clade and related to African Rhinotyphlops, forming a monophyletic group with R. newtoni and R. feae (bootsrap values = 100, pp = 1.0) (Fig. 2). It is worth noting that R. newtoni and R. feae, which occur on the volcanic island of São Tomé at the gulf of Guinea (Fig. 1), are also morphologically similar to the west Asian Rhinotyphlops (Roux-Estève, 1974). The estimated time of divergence between R. episcopus/R. simoni and R. newtoni/R. feae is in the Early Miocene, approximately 19.6 Mya (11.7–29.1 Mya). This time corresponds to the time of occurrence of their most recent common ancestor (MRCA). Unfortunately, no representatives of this particular subgroup from continental Africa are included in our analysis, and therefore two conclusions may be drawn: the estimated time of divergence corresponds to (a) the colonization of São Tomé by Rhinotyphlops, or (b) the split between sub-Saharan and Asian Rhinotyphlops. In the first scenario, the colonization of São Tomé by Rhinotyphlops occurred earlier than the isolation of Asian Rhinotyphlops from sub-Saharan ones (an event that cannot be dated). If this scenario is true, then Rhinotyphlops reached São Tomé through overseas dispersal at the very early stages of the island’s formation, since the oldest dated volcanic rocks are 15.7 My old (Deruelle et al., 1991). In the second scenario, it is the colonization
of São Tomé that cannot be dated, occurring some time later, but the isolation of R. simoni and R. episcopus from the African Rhinotyphlops occurred some 19.6 Mya (11.7–29.1 Mya), as a result of a possible vicarianistic event of geologic or climatic origin. Additionally, R. simoni and R. episcopus show a sister-species relationship to each other. R. simoni’s geographic distribution is restricted in Israel, Gaza strip, Golan Heights and western Jordan, while R. episcopus has a restricted distribution in southeastern Turkey (Fig. 1). There is a distributional gap of several hundreds of kilometers between them, while the distribution of R. simoni coincides to great extent with the distribution of T. vermicularis’ ‘‘clade A’’. Thus, the phylogenetic split of R. simoni/R. episcopus and the basal diversification within T. vermicularis seem to have occurred in the same region. Most importantly, they seem to have taken place during the same period, and specifically at 10.0 Mya (4.5– 16.8 Mya) and 9.2 Mya (3.8–16.0 Mya), respectively, implying the act of a common vicarianistic agent, i.e. the major aridification events that happened in the Tortonian (see above for T. vermicularis and ‘‘clade A’’). 3.3. Typhlops socotranus The Socotran archipelago rests on a shelf platform at the triple junction of the Gondwanan plates of India, Arabia and Africa. Tectonic activity in the Red Sea/Gulf of Aden region during the Oligocene marked the onset of the rifting between Arabia and Somalia continental blocks about 30–17.6 Mya (syn-rift) followed by a continental break-up and oceanic spreading at 17.6 Mya (post-rift) that increased the distance between the Socotra Archipelago and Arabian mainland (Autin et al., 2010). The complex geological history of the Socotran archipelago, together with its diverse topology and climate, are primary responsible for its high levels of endemism. Its herpetofauna includes 28 native and two introduced terrestrial species (Razzetti et al., 2011) but few of them have been investigated using appropriate dating methods. Although considered part of the Afrotropic ecoregion, most of its herpetofauna has Palearctic affinities. So far, it seems that Hemidactylus geckos, Trachylepis skinks and Mesalina lizards have colonized Socotra island from Arabia after its complete isolation (Gómez-Díaz et al., 2012; Sindaco et al., 2012; Joger and Mayer, 2002), while the snakes Hemerophis and Ditypophis and Chamaeleo chameleons were probably present on Socotra when it separated from mainland Arabia (Macey et al., 2008; Nagy et al., 2003). Our analyses show that T. socotranus is related to T. vermicularis, as they form a monophyletic group (bootstrap values = 100, pp = 1.0) (Fig. 2). This result was not anticipated since there is a distributional gap of >1500 km between these two species (>2500 between T. socotranus and T. vermicularis ‘‘clade A’’) (Fig. 1). On the other hand, the sea-barrier between Socotra and the Horn of Africa is only 250 km. Nevertheless, T. socotranus, along with T. vermicularis, lie within the Eurasia/Australasia major clade and not the African one. The time of divergence between T. socotranus and T. vermicularis is estimated to be 29.6 Mya (18.3–42.1 Mya), i.e. the age of their MRCA. This leads to two basic hypotheses for the underlying diversification event: (a) T. socotranus ancestor was isolated on Socotra at the very early stage of the island’s geological isolation (rifting between Arabia and Somalia began some 30 Mya) or (b) the T. vermicularis/T. socotranus ancestor had a much wider distribution in the past covering the Arabian peninsula and, a subsequent extinction event occurring approximately 29.6 Mya, acted as a vicariant agent and isolated populations in the north (T. vermicularis ancestor) and the south (T. socotranus ancestor). Palaeogeographical reconstructions do not seem to support the latter hypothesis (e.g. Popov et al., 2004), while the global climatic-cooling event
39
P. Kornilios et al. / Molecular Phylogenetics and Evolution 68 (2013) 35–41
occurring at the Eocene–Oligocene transition is a possible vicariant agent, as it is responsible for the mass extinctions of this era (Xiao et al., 2010 and references therein). Additionally, in this scenario, Socotra was colonized some time later, but the age of this colonization via overseas dispersal, cannot be definitively inferred. In any case, an extinction event has surely taken place at some point, since there are no typhlopids presently occurring in the Arabian Peninsula (Fig. 1).
3.4. Typhlopid dispersal to Eurasia A diversification event seems to have occurred within the Eurasia/Australasia clade some 61.1 Mya (51.3–70.7 Mya) splitting the western species (T. vermicularis and T. socotranus) from the Indo-Malayan and Australasian ones. According to Vidal et al. (2010), the Eurasian/Australasian group probably originated by a northward dispersal from Gondwana, either out of Africa through Europe and Asia (Laurasia) or out of India. The latter refers to a dispersal event from northeast Africa/Arabia to the drifting subcontinent of India (through land connections or a volcanic island arc) and finally to Asia after the collision of the Indian continent with Asia (see Chatterjee and Scotese, 2010). Both scenarios involve a single dispersal (colonization) event to Eurasia and a subsequent expansion westwards (even reaching Europe) and eastwards (reaching Australia), while the diversification between west and east Asian typhlopids can be explained by a vicarianistic event, occurring some 60 Mya. This event ‘‘broke-up’’ a continuous distribution into a western and an eastern part creating a distributional gap, that remains until today (Fig. 1). There are two problems concerning the hypotheses of a single dispersal (either from Africa or ‘‘out of India’’): (a) there is no known geologic or climatic event occurring at that time (Middle Paleocene) to have acted as a vicariant agent, and (b) especially for the ‘‘out of India’’ scenario, the diversification in east and west groups would have happened within India much earlier than the actual hard collision with Asia (35 Mya) (Ali and Aitchison, 2008). Although we do not reject these two hypotheses, we propose a third one that better fits our results. This involves two dispersal events from the same source, i.e. from north-east Africa/Arabia: one northwards-westwards which led to the west Asian typhlopids and a second one eastwards to India and later to south-east Asia, Working code
Species
Tv7 Rs1
T. vermicularis ‘‘Clade A’’ R. simoni
Re1
R. episcopus
Re2
R. episcopus
Ts1
T. socotranus
Ts2
T. socotranus T. T. T. T. T.
granti hypomethes platycephalus richardi naugus
Museum code
Locality (country)
NHMC 80.3.21.7 NHMC 80.3.21.8 MCCIR1619(1) MCCIR1619(2) MCCIR1432 MCCIR1493
leading to the east typhlopid group (‘‘out of India’’). This scenario explains (a) the monophyly (common ancestry) of Eurasian/Australasian Typhlopidae, (b) the distributional gap between them, since it involves two independent geographic expansions in the west and the east, respectively, and (c) it fits the estimated ages better. This hypothesis is also backed-up by the fact that the eastern part of the west-Asian typhlopids’ distribution (Fig. 1) is very recent (Middle or Late Pleistocene) (Kornilios et al., 2012), while the most morphologically similar species to T. vermicularis is the northwest African T. etheridgei (Wallach, 2002). Conclusively, blindsnakes (Typhlopoidea) have managed to disperse into Asia from Africa at least four times. A very early dispersal is described by the ‘‘out of India hypothesis’’ also known as the ‘‘Biotic ferry model’’. According to this, the rafting Indian plate carried ancient Gondwanan forms to Asia, now recognized as a separate family, the Gerrhopilidae (Vidal et al., 2010). During its ‘‘journey’’, a second dispersal occurred from northeast Africa/ Arabia to the rafting Indian subcontinent and subsequently to Asia after its collision. This led to the east Asia/Austalasia typhlopid radiation. A third dispersal from the same region to west Eurasia gave rise to the west Asia radiation (T. vermicularis species complex and T. socotranus). Finally, a forth dispersal from Africa to west Asia resulted in the asian Rhinotyphlops species.
Acknowledgments R.S. acknowledges for their help during field work Cristina Grieco and Elisa Riservato (Anatolia and Socotra), Fabio Pupin, Edoardo Razzetti and Mauro Fasola (Socotra).
Appendix A. Sample working codes, species names, museum numbers (NHMC: Natural History Museum of Crete; MCC: Museo Civico di Storia Naturale di Carmagnola, Italy) and sampling locations (locality/country) of the specimens analyzed in the current study. GenBank accession numbers of sequence data for the DNA segments are also shown (AMEL: amelogenin, BDNF: brain-derived neurotrophic factor, NT3: neurotrophin 3). GenBank accession number AMEL
BDNF
NT3
Zai Park (Jordan) 32.150037, 35.716553
KC848442
KC848451
KC848454
4 km S of Al Mazar, al Janubi (Jordan) 31.052934, 35.612183 S ß anliurfa prov., Halfeti env. (Turkey) 37.246181, 37.868156 S ß anliurfa prov., Halfeti env. (Turkey) 37.246181, 37.868156 Sokotra Isl., between Eerk and Jelhiiyo (Yemen) 12.567968, 54.162769 Sokotra Isl., Mala Plateau (Yemen) 12.655078, 53.491058
KC848447
KC848448
KC848459
KC848445
KC848449
KC848457
KC848446
KC848450
KC848458
KC848443
KC848452
KC848455
KC848444
KC848453
KC848456
GU902350 GU902351 GU902357 GU902358 GU902355
GU902430 GU902431 GU902437 GU902438 GU902435
GU902600 GU902601 GU902607 GU902608 GU902605
(continued on next page)
40
P. Kornilios et al. / Molecular Phylogenetics and Evolution 68 (2013) 35–41
(continued)
Working code
Species
T. catapontus T. dominicanus T. geotomus T. monastus T. caymanensis T. contorhinus T. anousius T. notorachius T. arator T. anchaurus T. rostellatus T. syntherus T. sulcatus T. capitulatus T. jamaicensis T. sylleptor T. agoralionis T. lumbricalis T. eperopeus T. schwartzi T. reticulatus T. brongersmianus T. angolensis T. obtusus T. elegans T. bibronii T. fornasinii T. lineolatus T. congestus T. punctatus R. schlegelii R. mucruso R. newtoni R. feae R. lalandei R. unitaeniatus T. luzonensis R. albiceps Ramphotyphlops sp. R. braminus T. pammeces R. lineatus R. acuticaudus Acutotyphlops sp. A. subocularis A. kunuaensis R. polygrammicus R. longissimus R. bituberculatus R. unguirostris R. kimberleyensis R. ganei R. ligatus R. bicolor R. splendidus R. pinguis R. hamatus R. australis R. pilbarensis R. waitii R. diversus
Museum code
Locality (country)
GenBank accession number AMEL
BDNF
NT3
GU902346 GU902348 GU902349 GU902354 GU902347 GU902366 GU902365 GU902356 GU902344 GU902343 GU902359 GU902363 GU902361 GU902345 GU902352 GU902362 GU902342 GU902353 GU902364 GU902360 GU902319 GU902313 GU902312 GU902375 GU902314 GU902370 GU902367 GU902371 GU902368 GU902318 GU902369 GU902310 GU902311 GU902308 GU902309 GU902372 GU902316 GU902305 GU902340 GU902306 GU902378 GU902307 GU902304 GU902379 GU902338 GU902339 GU902341 GU902330 GU902325 GU902329 GU902328 GU902334 GU902327 GU902332 GU902337 GU902336 GU902323 GU902331 GU902322 GU902324 GU902333
GU902426 GU902428 GU902429 GU902434 GU902427 GU902446 GU902445 GU902436 GU902424 GU902423 GU902439 GU902443 GU902441 GU902425 GU902432 GU902442 GU902422 GU902433 GU902444 GU902440 GU902396 GU902390 GU902389 – GU902314 GU902450 GU902617 GU902451 GU902448 GU902395 GU902449 GU902387 GU902388 GU902385 GU902386 GU902452 GU902393 GU902382 GU902420 GU902383 GU902458 GU902384 GU902381 GU902459 GU902418 GU902419 GU902421 GU902408 GU902403 GU902407 GU902406 GU902412 GU902405 GU902410 GU902416 GU902415 GU902401 GU902409 GU902400 GU902402 GU902411
GU902596 GU902598 GU902599 GU902604 GU902597 GU902616 GU902615 GU902606 GU902594 GU902593 GU902609 GU902613 GU902611 GU902595 GU902602 GU902612 GU902592 GU902603 GU902614 GU902610 GU902568 GU902563 GU902562 GU902625 GU902564 GU902620 GU902447 GU902621 GU902618 GU902567 GU902619 GU902560 GU902561 GU902558 GU902559 – – GU902555 – GU902556 GU902628 GU902557 GU902554 GU902629 GU902589 GU902590 GU902591 GU902579 GU902574 GU902578 GU902577 GU902583 GU902576 GU902581 GU902587 GU902586 GU902572 GU902580 GU902571 GU902573 GU902582
41
P. Kornilios et al. / Molecular Phylogenetics and Evolution 68 (2013) 35–41 (continued)
Working code
Species
Museum code
Locality (country)
R. howi R. guentheri T. arenarius T. andasibensis X. grandidieri G. mirus
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2013.03. 009. References Ali, J.R., Aitchison, J.C., 2008. Gondwana to Asia: plate tectonics, paleogeography and the biological connectivity of the Indian sub-continent from the Middle Jurassic through latest Eocene (166–35 Ma). Earth Sci. Rev. 88, 145–166. An, Z., Kutzbach, J.E., Prell, W.L., Porter, S.C., 2001. Evolution of Asian monsoons and phased uplift of the Himalaya-Tibetan Plateau since late Miocene times. Nature 411, 62–66. Autin, J., Leroy, S., Beslier, M.-O., d’Acremont, E., Razin, P., Ribodetti, A., Bellahsen, N., Robin, C., Al Toubi, K., 2010. Continental break-up history of a deep magmapoor margin based on seismic reflection data (northeastern Gulf of Aden margin, offshore Oman). Geophys. J. Int. 180, 501–519. Broadley, D.G., Wallach, V., 2007. A review of East and Central African species of Letheobia Cope, revived from the synonymy of Rhinotyphlops Fitzinger, with descriptions of five new species (Serpentes: Typhlopidae). Zootaxa 1515, 31–68. Chatterjee, S., Scotese, C., 2010. The wandering Indian plate and its changing biogeography during the late cretaceous-early tertiary period. In: Bandyopadhyay, S. (Ed.), New Aspects of Mesozoic Biodiversity, Lecture Notes in Earth Sciences. Springer, Berlin/Heidelberg, pp. 105–126. Das, I., Chaturvedi, N., 1998. Catalogue of herpetological types in the collection of the bombay Natural History Society. Hamadryad 23, 150–156. Datta-Roy, A., Karanth, K.P., 2009. The out-of-India hypothesis: what do molecules suggest? J. Biosci. 34, 687–697. Deruelle, B., Moreau, C., Nkoumbou, C., Kambou, R., Lissom, J., Njongfang, E., Ghogomu, R.T., Nono, A., 1991. The Cameroon Line: a review. In: Kampunzu, A.B., Lubala, R.T. (Eds.), Magmatism in Extensional Structural Settings. Springer Verlag, Berlin, Germany, pp. 274–327. Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214. Gómez-Díaz, E., Sindaco, R., Pupin, F., Fasola, M., Carranza, S., 2012. Origin and in situ diversification in Hemidactylus geckos of the Socotra Archipelago. Mol. Ecol. 21, 4074–4092. Graur, D., Martin, W., 2004. Reading the entrails of chickens: molecular timescales of evolution and the illusion of precision. Trends Genet. 20, 80–86. Hasegawa, M., Kishino, K., Yano, T., 1985. Dating the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22, 160–174. Joger, U., Mayer, W., 2002. A new species of Mesalina (Reptilia: Lacertidae) from Abd al-Kuri, Socotra Archipelago, Yemen, and a preliminary molecular phylogeny for the genus Mesalina. Fauna Arab. 19, 497–505. Kimura, M., 1981. Estimation of evolutionary distances between homologous nucleotide sequences. Proc. Natl. Acad. Sci. USA 78, 454–458. Kornilios, P., Ilgaz, Ç., Kumlutas, Y., Lymberakis, P., Moravec, J., Sindaco, R., RastegarPouyani, N., Afroosheh, M., Giokas, S., Fraguedakis-Tsolis, S., Chondropoulos, B., 2012. Neogene climatic oscillations shape the biogeography and evolutionary history of the Eurasian blindsnake. Mol. Phylogenet. Evol. 62, 856–873.
GenBank accession number AMEL
BDNF
NT3
GU902335 GU902326 GU902374 GU902373 GU902377 GU902317
GU902414 GU902404 GU902455 GU902453 GU902457 GU902394
GU902585 GU902575 GU902624 GU902622 GU902627 GU902566
Kuriyama, T., Brandley, M.C., Katayama, A., Mori, A., Honda, M., Hasegawa, M., 2011. A time-calibrated phylogenetic approach to assessing the phylogeography, colonization history and phenotypic evolution of snakes in the Japanese Izu Islands. J. Biogeogr. 38, 259–271. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948. Macey, J.R., Kuehl, J.V., Larson, A., Robinson, M.D., Ugurtas, I.H., Ananjeva, N.B., Rahman, H., Javed, H.I., Osman, R.M., Doummak, A., Papenfuss, T.J., 2008. Socotra Island the forgotten fragment of Gondwana: Unmasking chameleon lizard history with complete mitochondrial genomic data. Mol. Phylogenet. Evol. 49, 1015–1018. Nagy, Z.T., Joger, U., Wink, M., Glaw, F., Vences, M., 2003. Multiple colonization of Madagascar and Socotra by colubrid snakes: evidence from nuclear and mitochondrial gene phylogenies. Proc. R. Soc. Lond. B Biol. Sci. 270, 2613–2621. Popov, S.V., Rögl, F., Rozanov, A.Y., Steiniger, F.F., Shcherba, I.G., Kovac, M., 2004. Lithological-paleogeographic maps of Paratethys: 10 maps Late Eocene to Pliocene. Cour. Forsch.-Inst. Senckenberg 250, 1–46. Posada, D., 2008. JModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25, 1253–1256. Rambaut, A., Drummond, A.J., 2007. Tracer v1.5.