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Molecular phylogenetic analyses using mitochondrial COI gene sequences showed that Anodonta individuals from Nagoya and Toyohashi, Aichi Prefecture and Hokuto, Yamanashi Prefecture belong to Buldowskia shadini (Moskvicheva, 1973), proposing a new Japanese name “Yahazu-Numagai” for this species. This study raises the possibility that B. shadini individuals in various parts of Japan have been misidentified as Anemina arcaeformis (Heude, 1877) based on shell morphology.

Gyraulus soritai Habe, 1976 was discovered in a riparian wetland at Obora, Gifu City, Gifu Prefecture as the first record of this species from Gifu Prefecture. G. soritai is an endangered micromollusk and difficult to find in the wild. A detailed survey of this species will be expected at other wetlands of Gifu Prefecture.

Molecular phylogenetic analyses were conducted for Anodonta individuals from Okumikawa, Aichi Prefecture, Japan. Mitochondrial DNA sequences of these individuals were nearly identical to those of Buldowskia shadini that occurs from the Amur River basin in Russia, northeastern China, and Mongolia (Buir Lake) to South Korea. This is probably the first record of the species from Japan.

Stereophaedusa japonica is distributed in three areas of Gifu City, Gifu Prefecture: Akisawa and Oku near the northwestern end of the city, Miwa near the northeastern end, and Mizuburodani near the center of the city. DNA analyses showed that individuals from Mizuburodani and Miwa were identical or nearly identical to each other, while these sequences were more distantly related to those from Akisawa and Oku, suggesting that Mizuburodani populations derived from Miwa populations by migration and settlement via rivers.

Read numbers obtained using the polyA_seq.pl program in different conditions. Figure S2. Minor polyadenylation sites for T. tachydromoides mitochondrial RNAs. Figure S3. Confirmation of the new polyadenylation site for T. tachydromoides ND5 mRNA by 3′ RACE. Figure S4. 5′ end mapping of T. tachydromoides cDNA fragments with polyadenylation at their 3′ end inside mitochondrial tRNA genes. Figure S5. Major polyadenylation sites for 11 species in which the polyadenylation profile was similar to that of the human. Figure S6. Characterization of E. macularius mt-mRNAs using RNA-Seq reads. Figure S7. Characterization of P. castaneus mt-mRNAs using RNA-Seq reads. Figure S8. Characterization of A. mississippiensis mt-mRNAs using RNA-Seq reads. Figure S9. Characterization of Homo sapiens mt-mRNAs using directional RNA-Seq reads (SRR3151753). Figure S10. The secondary structure of the L-strand sequence around the de novo polyadenylation site for the T. tachydromoides ND5 mRNA. (PDF 1437 kb)

Molecular phylogenic trees of WAC gene. This figure shows neighbor-joining trees of WAC gene with the long alignment for 21 tetrapod species and the short alignment for 21 squamate species. (PDF 282 kb)

Diagrams for partial gene structure, primer positions and gel electrophoresis of PCR products. This file provides information about primer positions on CTNNB1 and WAC genes, and images of gel electrophoreses for PCR amplicons of partial sequences of CTNNB1 gene in snakes. (PDF 401 kb)

Alignment of full-length amino acid sequences of CTNNB1 among tetrapod species. This figure shows alignment of full-length amino acid sequences of CTNNB1 among 11 tetrapod species. (PDF 306 kb)

Alignment of full-length amino acid sequences of WAC among tetrapod species. This figure shows alignment of full-length amino acid sequences of WAC among 11 tetrapod species. (PDF 302 kb)

CLASSIFICATION OF UROMASTYX We discuss the validity of the morphology-based classification of Uromastyx in the light of their molecular phylogeny. The molecular data support Uromastyx and Leiolepis forming a sister-group relationship as members of the most basal agamid lineage (57–88% bootstrap values; Fig. 5). Morphological studies since that conducted by Moody (1980) have pointed to this relationship, classifying them into a separate family Uromastycidae (Böhme, 1982), subfamily Uromastycinae (Borsuk-Bialynicka & Moody, 1984) or Leiolepidinae (Frost & Etheridge, 1989; Ananjeva, Dujsebayeva & Joger, 2001). To the best of our knowledge, molecu- lar studies have not provided sufficient resolution on this matter. Partial 12S or 16S rRNA gene sequences supported the sister relationship of Uromastyx and Leiolepis, but did not support the view that the clade represents one of the earliest agamid lineages (Honda et al., 2000). A tree constructed by Macey et al. (2000) using gene regions similar to ours did not even support the clustering of the two genera, although their taxonomic representations were somewhat biased towards agamids other than Uromastyx and Leiolepis. Therefore, there still seems to be uncertainty in the placement of Uromastyx and Leiolepis in the Agamidae, and this should be investigated further with increased molecular data. The molecular phylogeny within the Uromastyx species (Fig. 5) is in good agreement generally with the most recent view based on morphology (Wilms, 2001). U. hardwickii represents the most basal lineage of the genus and the remaining taxa investigated grouped into the African and Arabian clades. However, as outlined earlier, there were a few discrepancies with respect to the phylogenetic affiliation of U. macfadyeni, U. aegyptia and U. benti. Morphological studies (Moody, 1987; Wilms, 2001) pointed to certain similarities of U. macfadyeni to U. ocellata and U. ornata of the Arabian clade, with the last 12–21 tail whorls made up of a continuous scale row and with fewer than 260 scales around the midbody. In order to examine this further, we conducted the Kishino–Hasegawa test to compare the log-likelihood values between the ML tree topology of Figure 5 and some competing hypotheses in which U. macfadyeni clusters with either or both U. ocellata and U. ornata (Table 3). The results clearly rejected the competing hypotheses at the 1% significance level. We therefore suppose that the apparent similarities between U. macfadyeni and the latter two species may be due to convergent evolution through sharing similar ecological or climatic environments. These species are now distributed in neighbouring areas surrounding the Red Sea and the Gulf of Aden (Fig. 6). The nucleotide sequences (1503 bp) for the 25 taxa were analysed as described in the Material and methods. User-defined unrooted tree topologies were as follows: tree 1 for the ML tree topology as shown in Fig. 5, (Outgroup, Hard, ((Mac, (Gey, ((Aca1, Aca2), (Dis, Mali)))), ((Aeg, Mic), (Ben, ((Oce1, Oce2), Orn))))); tree 2, (Outgroup, Hard, ((Gey, ((Aca1, Aca2), (Dis, Mali))), ((Aeg, Mic), (Ben, (Mac, ((Oce1, Oce2), Orn)))))); tree 3, (Outgroup, Hard, ((Gey, ((Aca1, Aca2), (Dis, Mali))), ((Aeg, Mic), (Ben, (Orn, ((Oce1, Oce2), Mac)))))); and tree 4, (Outgroup, Hard, ((Gey, ((Aca1, Aca2), (Dis, Mali))), ((Aeg, Mic), (Ben, ((Oce1, Oce2), (Mac, Orn)))))). Refer to Fig. 5 for the tree topology of the outgroup and see the legend of Fig. 6 for abbreviations of Uromastyx taxa. Oce1 and Oce2 mean U. ocellata from Egypt and Sudan, respectively. †Natural logarithm of the likelihood value. ‡Difference in lnL from that of the ML tree. §A standard error in lnL. ¶An asterisk means that the corresponding phylogenetic hypothesis can be statistically rejected at the 1% significance level by the standard criterion of DlnL/SE> 2.58. U. aegyptia was considered to have a phylogenetic affinity to the African group because of their common features in external morphology, such as the last two to five tail whorls being made up of a continuous scale row (Moody, 1987; Wilms, 2001). However, this species has much higher midbody scale counts than do members of the African group (Wilms & Böhme, 2000a). The phylogenetic affiliation of U. aegyptia in the Arabian group (Fig. 5) may therefore be possible, though this is not conclusive due to the low bootstrap values (52–57%). It is noteworthy that the serological studies by Joger (1986) also suggested a closer relationship of U. aegyptia to U. ocellata and U. ornata than to U. acanthinura and U. geyri. Finally, the molecular phylogeny (Fig. 5) placed U. benti as sister to the U. ocellata – U. ornata clade, while U. benti was morphologically regarded as a sister taxon of U. ocellata with the exclusion of U. ornata (Wilms, 2001). We favour the molecular view in this respect because of its strong bootstrap values (98–99%). Treatment of a taxon as either a species or a subspecies has been changed frequently for members of the genus Uromastyx, and this may still be controversial. For example, Mertens (1962) classified U. geyri and U. dispar as subspecies of U. acanthinura, while Moody (1980) recognized them as full species. Thomas Wilms followed the former view in his book (Wilms, 1995) but later changed to the latter (Wilms, 2001). He also once recognized U. ocellata, U. ornata and U. macfadyeni as subspecies of U. ocellata (Wilms, 1995), while all of them have recently been treated as independent species (Wilms & Böhme, 2000c; Wilms, 2001). Conversely, U. d. maliensis, once recognized as an independent species (Joger & Lambert, 1996), has been revised to be a subspecies of U. dispar (Wilms & Böhme, 2000b). In all the above-mentioned points, our molecular phylogeny (Fig. 5) supports the most recent view in Wilms (2001), not only for the phylogenetic relationship but also for the level of divergence. The divergence times between U. geyri, U. acanthinura and U. dispar, as well as between U. ocellata and U. ornata, were much larger compared with those between U. d. dispar and U. d. maliensis (Table 2). HISTORICAL BIOGEOGRAPHY Extant Uromastyx taxa of the African and Arabian groups are distributed allopatrically, with a considerable overlap of distribution only between U. geyri and U. d. maliensis (Fig. 6). In this section, we discuss how the Uromastyx species radiated and migrated to their current habitats, based on our molecular data together with geological and palaeoenvironmental evidence. The evolutionary framework thus constructed using representative sequences from each taxon will provide a basis for future phylogeographical analyses using many individuals with detailed locality information. Our molecular analyses suggested that Uromastyx and Leiolepis are sister genera (Fig. 5) and that they diverged from each other in the middle Eocene (40–50 Mya; Table 2). The oldest fossil record closely associated with these genera is Mimeosaurus of the Upper Cretaceous–Eocene of Mongolia (Moody, 1980), and is consistent with the above-mentioned divergence time. Moreover, all the extant species included in the genus Leiolepis inhabit south-east Asia. U. hardwickii, having diverged from the most basal position of the Uromastyx phylogeny (Fig. 5), inhabits south Asia. Thus, it is likely that direct ancestors of these genera lived in central–south Asia, from where the genus Uromastyx originated and migrated westward towards the hot and arid habitats suitable for their lifestyle (Fig. 6). From the late Cretaceous to the early Miocene (18–100 Mya) the African continent, including an area of the current Arabian Peninsula, had long been isolated from other continents (Rögl, 1998). Geological and palaeontological evidence (Rögl, 1998; Harzhauser, Piller & Steininger, 2002) consistently shows that plate tectonic activities connected Africa to Eurasia through closure of the Eastern Mediterranean seaway by approximately 18 Mya (the Gomphotherium Landbridge). This landbridge later became disconnected temporarily, but it has been continuously present since ~15 Mya. Our molecular analyses suggested that the Asian (U. hardwickii) and Afro-Arabian (the other species used) taxa diverged 25–29 Mya (Table 2). This is much earlier compared with the estimate for the formation of the Gomphotherium Landbridge. We therefore speculate that there was an initial stage of radiation of the Uromastyx lizards in the eastern Middle East before the formation of the landbridge, and that predecessors of the Afro-Arabian taxa derived from one of these diversified lineages. A few taxa from the eastern Middle East (e.g. U. loricata of Iraq and Iran and U. thomasi of Oman) were not included in our study. Morphological (Wilms, 2001 and refs. therein) and immunological (Joger, 1986) studies have suggested that these taxa diverged from basal positions next to that of U. hardwickii in the Uromastyx phylogeny. We estimated the African and Arabian groups of Uromastyx to have diverged 11–15 Mya (Table 2). North Africa was subjected to climatic changes towards aridity in the middle Miocene. Dry and open woodlands and the savanna emerged to interrupt the continuous African forest by the late middle Miocene (McClanahan & Young, 1996; MacDonald, 2003), and this may have facilitated faunal and floral exchanges eastwards and westwards (e.g. Fu, 1998; Caujapé- Castells & Jansen, 2003). Predecessors of the African Uromastyx group may have migrated to new xeric habitats in North Africa and diverged from the Arabian taxa (Fig. 6). The Arabian Peninsula began to separate from the remaining part of the African continent in the early Miocene as a tectonic consequence of the Afar mantle plume leading to the formation of the Red Sea, the Gulf of Aden and the East African Rift Valley (Girdler, 1991; Pudlo, Shandelmeier & Reynolds, 1997). This accompanied considerable uplifting of some mountain systems along the plate boundaries. The East African Rift Valley and associated mountain systems may have become terrestrial barriers, isolating U. macfadyeni of northern Somalia from its sister taxa of the African group. The estimated divergence time (10–12 Mya; Table 2) seems consistent with geological (Girdler, 1991) and palaeontological (Coppens, 1994) data in supporting this idea. Within the Arabian group, U. aegyptia first diverged from the other members 12–14 Mya (Table 2) and this species is now widely distributed in the Arabian Peninsula and northern Egypt. Molecular data also suggested that U. benti diverged from U. ocellata and U. ornata 9–10 Mya and that the latter two species diverged from each other 7–8 Mya (Table 2). A possible geological factor that may have been associated with the former divergence is the elevation of the Yemen Plateau (Geoffroy, Huchon & Khanbari, 1998). However, the considerable uplifting of the Yemen Plateau may have already occurred in the early Miocene, somewhat earlier than the estimated divergence time (9–10 Mya). We therefore withhold a conclusion on this matter until more molecular and geological data become available. Geological influence on the divergence between U. ocellata and U. ornata seems much clearer. Since the current distribution ranges for U. ocellata and U. ornata are separated by the Red Sea, their divergence has been hypothesized to be due to the habitat fragmentation caused by the expansion of the Red Sea (Wilms, 2001). Our study supports this hypothesis by showing that the estimated divergence time between the two species (7–8 Mya) corresponds well to the geological timing for the expansion of the Red Sea (Girdler, 1991; Pudlo et al., 1997; refs. therein). The oceanic accretion may have started in the middle Miocene (12– 13 Mya), while evidence from sedimentary rocks suggests that seawater had come in to the northern region of the Red Sea by at least 5 Mya (Ross & Schlee, 1973). Our molecular data pointed to much more recent times for the divergences between members of the African group other than U. macfadyeni (Table 2). By the early Pliocene (around 5 Mya), the trend for a cooler and drier environment was well established in North Africa with expansion of the grassland (MacDonald, 2003). After 2.8 Mya, there were repeated global cycles of warming and cooling and this is believed to have accelerated speciation for a variety of terrestrial and marine animals (Agusti, Rook & Andrews, 1999; deMenocal & Brown, 1999). Especially during the late Pliocene (around 2.4 and 1.8 Mya), further cooling and drying resulted in a major expansion of grassland and desert environments (McClanahan & Young, 1996). We suggest that such climatic fluctuations could have caused the habitat fragmentation and isolation of local populations, leading to the speciation between U. geyri, U. acanthinura and U. dispar., Published as part of Amer, Sayed A. M. & Kumazawa, Yoshinori, 2005, Mitochondrial DNA sequences of the Afro-Arabian spiny-tailed lizards (genus Uromastyx; family Agamidae): phylogenetic analyses and evolution of gene arrangements, pp. 247-260 in Biological Journal of the Linnean Society 85 on pages 254-257, DOI: 10.1111/j.1095-8312.2005.00485.x, http://zenodo.org/record/7846312, {"references":["Moody SM. 1980. Phylogenetic and historical biogeographical relationships of the genera in family Agamidae (Reptilia: Lacertilia). PhD Thesis, University of Michigan, Ann Arbor, Michigan.","Bohme W. 1982. Uber Schmetterlingsagamen, Leiolepis belliana belliana, der malayischen Halbinsel und ihre parthenogenetischen Linien (Sauria, Uromastycidae). 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Amer, Sayed A. M., Kumazawa, Yoshinori (2005): Mitochondrial DNA sequences of the Afro-Arabian spiny-tailed lizards (genus Uromastyx; family Agamidae): phylogenetic analyses and evolution of gene arrangements. Biological Journal of the Linnean Society 85: 247-260, DOI: 10.1111/j.1095-8312.2005.00485.x