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New evidence on the phylogenetic position of the poorly known Asian pitviper Protobothrops kaulbacki (Serpentes: Viperidae: Crotalinae) with a redescription of the species and a revision of the genus Protobothrops

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Although much systematic work has been done in recent years on the Asian pitviper genus Protobothrops, the phylogenetic position of P. kaulbacki remains poorly understood due to its rarity and the inaccessibility of its range. This species has long been regarded as morphologically close to P. jerdonii and therefore has been widely treated as a member of Protobothrops. In this paper, we evaluate the phylogenetic position of this species using skull characteristics, hemipenial, ecological and molecular data. A molecular phylogeny, based on four mitochondrial genes, shows that the species forms a very highly supported sister-group relationship with Triceratolepidophis sieversorum, and is distinct from all other Protobothrops species. We discuss the alternative systematic arrangements that could take into account these newly discovered relationships of P. kaulbacki, provide a redescription of the species and summarize the available information on the distribution and natural history of P. kaulbacki.
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... The origins of toxin ratios are marked alphabetically: a [37], b [31], c and d [50]. The phylogentic relationship is based on [15,51]. ...
... mucrosquamatus), named the Taiwan Habu, as well as the Trimeresurus stejnegeri (T. stejnegeri) (indicated with P3, P4 in Figure 4) [15,51]. As for P. flavoviridis, they can be found on island habitats, e.g., Taiwan, in the south-west of Okinawa at the end of the Japanese island chain, and the P. mucrosquamatus was also observed directly on Okinawa [15,51,52]. ...
... stejnegeri) (indicated with P3, P4 in Figure 4) [15,51]. As for P. flavoviridis, they can be found on island habitats, e.g., Taiwan, in the south-west of Okinawa at the end of the Japanese island chain, and the P. mucrosquamatus was also observed directly on Okinawa [15,51,52]. Like the Habu from Japan, these two vipers belong to the medically important venomous snakes in their Taiwanese habitat and are responsible for significant envenomations and deaths over the last decades [53,54]. ...
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The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA2). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA2s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), L-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5′-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.
... research in recent years, with numerous new taxa being formally described and proposals made to split the genus as recognized to smaller divisions. Notable recently published studies on the systematics of these snakes include those of Das and Yaakob (2007), David, et. al. (2006), David, et. al. (2011), Gumprecht (1998), Gumprecht, et. al. (2004), Guo, et. al. (2007), Guo, et. al. (2009), Guo and Wang, (2011), Malhotra and Thorpe (2004), Malhorta, et. al. (2011), McDiarmid, et. al., (1999, Tu et al. (2000) and Zhao and Adler (1993). Recent divisions within Trimeresurus senso lato or reinterpretations of the genus, has resulted in the transfer of species to the following genera: Garthius Das and Yaak ...
... e subject genera and to stabilize the nomenclature. Ceratrimeresurus Liang and Liu, (2003) was synonymised with Protobothrops in 2008 (David et. al. 2008). Ermia Zhang, 1993 is not an available name for snake taxa (already a genus name for something else) and Zhaoermia Gumprecht and Tillack, 2004 was later found to be synonymous with Protobothrops (Guo et. al. 2007). Within the genus Cryptelytrops as recently resurrected, there is a distinct division between two main groups. About half the described species including the type species, C. purpureomaculatus are clearly very similar. However six species have been known to be divergent, these being the species formally described as "Cryptelytrops carda ...
Article
The Asian Pitvipers, recently placed in the genus genus Cryptelytrops Cope, 1860, have long been recognized as a distinct group. Recently a number of phylogentic studies including Pyron et. al. (2011) and Malhorta et. al. (2011) have confirmed simple observations of morphology to show that within this assemblage of about a dozen described species, six species of these snakes differ significantly from other members of the genus Cryptelytrops senso lato. The type species for the genus Cryptelytrops is the morphologically distinct taxa C. purpureomaculatus. This paper formalizes the obvious phylogenetic divergence by placing the six divergent species in a new genus according to the current Zoological Code (Ride et. al. 1999). Placed in Adelynhoserea gen. nov. are the species formerly placed in the genus Cryptelytrops, namely C. cardamomensis, C. Honsonensis, C. kanburiensis, C. macrops, C. rubeus and C. venustus.
... Notable published studies of the systematics of these snakes include those of Das and Yaakob (2007), David, et. al. (2011), Gumprecht, et. al. (2004), Guo, et. al. (2007), Guo, et. al. (2009), Guo and Wang, (2011), Malhotra and Thorpe (2004), Malhorta, et. al. (2011), McDiarmid, et. al., (1999), Tu, et al. (2000 and Zhao and Adler (1993). ...
... Ceratrimeresurus Liang and Liu (2003) was synonymised with Protobothrops in 2008 (David et. al. 2008). Ermia Zhang 1993 is not an available name for snake taxa (already a genus name for something else) and Zhaoermia Gumprecht and Tillack 2004 was later found to be synonymous with Protobothrops (Guo et. al. 2007). ...
Article
This paper formally recognises the Taiwan Mountain Pitviper Trimeresurus gracilis Oshima, 1920 as being sufficiently distinct from similar pitvipers of the genus Trimeresurus Lacépède, 1804 (type species T. viridis) to be placed in a different genus. While several other genera have been created to accommodate species previously placed within or likely to be placed within Trimeresurus sensu lato, the taxon Trimeresurus gracilis Oshima, 1920 does not fit within any. Therefore this paper formally names and diagnoses a new genus Oxyus gen. nov. to accommodate this taxon. At the present time this is a monotypic genus. Keywords: new genus; Trimeresurus; gracilis; Oxyus; Viperidae; Crotalinae; Hoser; snake; genus.
... The system of the family Viperidae remains controversial (Tu et al., 2000;Malhotra and Thorpe, 2000;Castoe and Parkinson, 2006;Creer et al., 2006;Guo et al., 2007;Wüster et al., 2008). During the last 10 -15 years numerous phylogenetic analyses based on molecular data were conducted, and their results are often conflicting. ...
... The species Zhaoermia mangshanensis (Zhao, 1990) was referred to the genus Protobothrops. The last fact is confirmed by molecular studies of Guo et al. (2007) as well as data on the position of Triceratolepidophis sieversorum Ziegler, Herrmann, David, Orlov et Pauwels, 2000 in the genus Protobothrops. According to data of Guo and Zhao (2006), the close relation of the species Zhaoermia mangshanensis to snakes of the genus Protobothrops is confirmed by skull morphology. ...
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The system of the family Viperidae remains controversial despite the fact that numerous studies had place during last years. The results of different analyses most of which are based on molecular data are conflicting. We conducted the phylogenetic analysis including 39 osteological characters and 31 species of Old World viperid snakes. According to these results, genera Azemiops and Causus do not belong to subfamily Viperinae, so their separation into distinct subfamilies is supported. The validity of subfamily Azemiopinae is confirmed by molecular data. The common primitive character of Azemiops and Causus is joining of the prefrontals to the lateral parts of the frontals, while in the true vipers (Viperinae) the prefrontals join to the anterior margins of the frontals and the contact between these bones nearly reaches the midline of the skull. This type of articulation and the particular shape of the prefrontal can be added to the diagnosis of subfamily Viperinae (without Azemiops and Causus). One more result of our analysis is that the most basal form among true vipers is genus Cerastes. Other true vipers are divided into two groups. Inside the first group, including genera Atheris, Bitis, Echis, and Vipera, the genera Atheris and Bitis form a clade what is consistent with results of some molecular studies. The second group includes genera Daboia, Macrovipera, Montivipera, and Pseudocerastes. In subfamily Crotalinae the clade including genera Cal-loselasma and Hypnale is strongly supported, that confirms the results of many molecular studies. Protobothrops mangshanensis is close to two other species of genus Protobothrops that is not in conflict with referring P. mang-shanensis (originally Zhaoermia mangshanensis) to this genus.
... The Old World pit viper genus Protobothrops is recognized as widely distributed among other Asian genera including Cryptelytrops, Garthius, Himalayophis, Ovophis, Parias, Peltopelor, Popeia, Trimeresurus, and Viridovipera [1][2][3]. The newly described Protobothrops kelomohy from Chiang Mai and Tak Provinces, Thailand [4], is one of 15 species currently validated in the genus Protobothrops, which are P. cornutus, P. dabieshanensis, P. elegans, P. flavoviridis, P. himalayanus, P. jerdonii, P. kaulbacki, P. manshenensis, P. maolanensis, P. mucosquamatus, P. sieversorum, P. tokaensis, P. trungkhanensis and P. xiangchengensis [5,6]. ...
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Background: A new pit viper, Protobothrops kelomohy, has been recently discovered in northern and northwestern Thailand. Envenoming by the other Protobothrops species across several Asian countries has been a serious health problem since their venom is highly hematotoxic. However, the management of P. kelomohy bites is required as no specific antivenom is available. This study aimed to investigate the biochemical properties and proteomes of P. kelomohy venom (PKV), including the cross-neutralization to its lethality with antivenoms available in Thailand. Methods: PKV was evaluated for its neutralizing capacity (ER50), lethality (LD50), procoagulant and hemorrhagic effects with three monovalent antivenoms (TAAV, DSAV, and CRAV) and one polyvalent (HPAV) hematotoxic antivenom. The enzymatic activities were examined in comparison with venoms of Trimeresurus albolabris (TAV), Daboia siamensis (DSV), Calloselasma rhodostoma (CRV). Molecular mass was separated on SDS-PAGE, then the specific proteins were determined by western blotting. The venom protein classification was analyzed using mass spectrometry-based proteomics. Results: Intravenous LD50 of PKV was 0.67 µg/g. ER50 of HPAV, DSAV and TAAV neutralize PKV at 1.02, 0.36 and 0.12 mg/mL, respectively. PKV exhibited procoagulant effect with a minimal coagulation dose of 12.5 ± 0.016 µg/mL and hemorrhagic effect with a minimal hemorrhagic dose of 1.20 ± 0.71 µg/mouse. HPAV was significantly effective in neutralizing procoagulant and hemorrhagic effects of PKV than those of TAAV, DSAV and CRAV. All enzymatic activities among four venoms exhibited significant differences. PKV proteome revealed eleven classes of putative snake venom proteins, predominantly metalloproteinase (40.85%), serine protease (29.93%), and phospholipase A2 (15.49%). Conclusions: Enzymatic activities of PKV are similarly related to other viperid venoms in this study by quantitatively hematotoxic properties. Three major venom toxins were responsible for coagulopathy in PKV envenomation. The antivenom HPAV was considered effective in neutralizing the lethality, procoagulant and hemorrhagic effects of PKV.
... In recent decades, species taxonomy has developed rapidly in large part from the use of molecular data, resulting in changes in nomenclature of animals included on protected species lists, which affects protection and management objectives [25]. Some changes are a simple name change based on new phylogenetic information such as the Chinese Strip-necked Turtle 'Ocadia sinensis' changing to Mauremys sinensis [26] or the Mangshan Pit Viper 'Zhaoermia mangshanensis' changing to Protobothrops mangshanensis [27]. Nomenclatural changes also occur when a polymorprhic species is divided into multiple species. ...
Article
China has about 11% of the world’s total wildlife species, so strengthening China’s wildlife conservation is of great significance to global biodiversity. Despite some successful cases and conservation efforts, 21.4% of China’s vertebrate species are threatened by human activities. The booming wildlife trade in China has posed serious threat to wildlife in China and throughout the world, while leading to a high risk of transmission of infectious zoonotic diseases. China’s wildlife conservation has faced a series of challenges, two of which are an impractical, separated management of wildlife and outdated protected species lists. Although the Wildlife Protection Law of China was revised in 2016, the issues of separated management remain, and the protected species lists are still not adequately revised. These issues have led to inefficient and overlapping management, waste of administrative resources, and serious obstacles to wildlife protection. In this article, we analyze the negative effects of current separated management of wildlife species and outdated protected species lists, and provide some suggestions for amendment of the laws and reform of wildlife management system.
... relied upon include: Beaman and Hayes (2008), Bocourt (1868), Boulenger (1888Boulenger ( , 1890Boulenger ( , 1892Boulenger ( , 1896, Boulenger et al. (1907), Boundy (2007), Bourret (1934), Broadley (1996), Bryson et al. (2011), Campbell and Lamar (2004), Campbell and Smith (2000), Carrasco et al. (2009), Castoe and Parkinson (2006), Castoe et al. (2005), Cope (1887), David (1995), David and Tong (1997), David and Vogel (1998, 2012), David et al. (2001, 2002a, 2002b, 2006, 2008, 2011), De Rooij (1917, Duméril et al. (1854), Fenwick, et al. (2009), Fernandes (2005, Fernandes et al. (2004), Garman (1881), Garrigues et al. (2005), Gloyd and Conant (1989), Gong et al. (2011), Grismer et al. (2006, , Groombridge (1986), , , Günther (1864), Guo et al. (1999aGuo et al. ( , 1999bGuo et al. ( , 2006Guo et al. ( , 2007Guo et al. ( , 2009), Gutberlet and Campbell (2001), Harvey (1994), Heise et. al. (1995, Herrmann et al. (1992Herrmann et al. ( , 2002, Romano-Hoge (1981, 1983), Hoser (2012aHoser ( , 2012bHoser ( , 2012cHoser ( , 2012dHoser ( , 2012e, 2012f, 2012g, 2012h, 2013aHoser ( , 2013bHoser ( , 2013c, Ineich, et al. (2006), Isogawa et al. (1994), Jadin et al. (2010Jadin et al. ( , 2011, Jan (1859), Jiang and Zhao (2009), Kardong (1986), Kelly et al. (2003), Klauber (1972), Koch (2008), Kraus, et al. (1996), Kuch et al. (2007), Lawson (1977), Lenk et. ...
Article
The generic arrangement of the Vipers has been subject of considerable change in recent years. The majority of reviews in the period 1990-2013 have tended to divide formerly large genera along phylogenetic lines. Most recently erected genera have had widespread acceptance within the herpetological community. A review of the Viperidae has shown inconsistent treatment of species groups, with some accorded recognition at the genus level, while others of similar divergence remain subsumed within larger paraphyletic genera. In order to make the treatment of Viper species at the genus level consistent, a review was undertaken including checking all relevant published literature, descriptions and phylogenies as well as direct inspection of specimens, including live, photos and museum specimens. As a result of earlier published papers by myself (including a paper published simultaneously to this one) (Hoser 2013c) and papers by others, the taxonomy and nomenclature of the True Vipers (Viperinae) appears to be consistent, based on this review. However, within the Pitvipers a very different picture emerged with several groups (clades) requiring formal taxonomic recognition at the genus or subgenus level. This was most notably the case for the deeply divergent and morphologically convergent Asian taxa. As a result, these unnamed groups are formally described for the first time, according to the Zoological Code (Ride et al. 1999). All groups are named on the basis of robust morphological and molecular data (refer to Hoser 2013b) and as identified in this paper. These are 8 newly named genera and 8 newly named subgenera. At the subfamily level, two morphologically divergent Tribes, namely Calloselasmiini Hoser, 2013 (Hoser 2013a) and Tropidolaemusini Hoser, 2012 are each placed in newly defined subfamilies on the basis of recent phylogenetic studies and published results which shows their continued placement within Crotalinae to be problematic. An updated list of Viper subfamilies, tribes and genera is presented. Keywords: Taxonomy; Pitvipers; new subfamilies; Tropidolaemusiinae; Calloselasmiinae; new genera; Sloppvipera; Conantvipera; Katrinahoserviperea; Ninvipera; Ryukyuvipera; Cummingviperea; Crottyvipera; Swilevipera; new subgenera; Blackleyviperea; Pughvipera; Davievipera; Cottonvipera; Lowryvipera; Simpsonvipera; Yunnanvipera; Borneovipera.
... and opinions relied upon include: Beaman and Hayes (2008), Bryson, et. al. (2011), Campbell and Smith (2000), Castoe and Parkinson (2006), David et. al. (2002), Dawson, et. al. (2008), Fernandes (2005), Garrigues et. al. (2005), Gloyd and Conant (1989), Fernandes et. al. (2004), Grismer et. al. (2006), Gumprecht et. al. (2004), Guo et. al. (1999), Guo et. al. (2007), Guo et. al. (2009), Jadin et. al. (2010, Jadin, et. al. (2011), Klauber, L. M. (1972), Kraus, et. al. (1996), , McCranie (2011), McDairmid et. al. (1999, Meik andPires-daSilva (2009), Pitman (1974), Smith (1941), Vogel (2006), Werman (1984), , Wüster and Bérnils (2011), Zamudio and Green (1997). ...
Article
This paper reviews recent phylogenetic studies of the Vipers to revisit the higher taxonomy of the group, specifically with reference to the level between family and genus. Three subfamilies Azemiopine, Crotalinae and Viperinae are recognised. The various tribes are redefined, diagnosed and named when there are no pre-existing valid names as determined by the ICZN rules current from year 2000. As a result, a total of 16 tribes are herein formally defined and named, many of them new. For the Azemiopine, one previously named tribe is identified. For the Crotalinae a total of 7 tribes are named and defined, 5 new, as well as several new subtribes. For the Viperinae a total of 8 tribes are named and defined, 5 new, as well as several new subtribes.
... In the present study, we examined the relative contributions of adaptive selection and genetic drift to evolution of venom and housekeeping genes, the latter defined as genes expressed in the venom gland, but not secreted into the venom proteome. We focused on the elaborated venom system of a pitviper, the Taiwan habu (Protobothrops mucrosquamatus), comparing it with its sister species, the Sakishima habu (Protobothrops elegans), from which it split approximately 2.9 Ma (Guo et al. 2007;Hedges et al. 2015). We find that both positive selection and genetic drift play a role in the rapid sequence changes seen in venom genes. ...
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Venoms are among the most biologically active secretions known, and are commonly believed to evolve under extreme positive selection. Many venom gene families, however, have undergone duplication, and are often deployed in doses vastly exceeding the LD50 for most prey species, which should reduce the strength of positive selection. Here we contrast these selective regimes using snake venoms, which consist of rapidly-evolving protein formulations. Though decades of extensive studies have found that snake venom proteins are subject to strong positive selection, the greater action of drift has been hypothesized, but never tested. Using a combination of de novo genome sequencing, population genomics, transcriptomics, and proteomics, we compare the two modes of evolution in the pitviper, Protobothrops mucrosquamatus. By partitioning selective constraints and adaptive evolution in a McDonald-Kreitman-type framework, we find support for both hypotheses: venom proteins indeed experience both stronger positive selection, and lower selective constraint than other genes in the genome. Furthermore, the strength of selection may be modulated by expression level, with more abundant proteins experiencing weaker selective constraint, leading to the accumulation of more deleterious mutations. These findings show that snake venoms evolve by a combination of adaptive and neutral mechanisms, both of which explain their extraordinarily high rates of molecular evolution. In addition to positive selection, which optimizes efficacy of the venom in the short term, relaxed selective constraints for deleterious mutations can lead to more rapid turnover of individual proteins, and potentially to exploration of a larger venom phenotypic space.
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DAVID P. & INEICH I., 1999 - Les serpents venimeux du monde: systématique et répartition. Dumerilia, Paris, 3: 3-499 [ouvrage publié avec le soutien financier des Laboratoires PASTEUR-MERIEUX, Lyon].
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— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.