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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
Abstract and Figures
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]. ...
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.
... However, as the nomen for the genus Ermia was already in use for a genus of locusts and was preoccupied, Gumprecht and Tillack (2004) provided a replacement name for this taxon and the species was assigned to a monotypic genus Zhaoermia. The first molecular phylogenetic data on these snakes, provided by Guo et al. (2007), indicated close relationships between Zhaoermia and Triceratolepidophis and Protobothrops and both former genera were coined as subjective junior synonyms of Protobothrops (Guo et al. 2007). Protobothrops mangshanensis is one of the largest members of the genus and is clearly different from other congeners in both external morphology, colouration and skull morphology (see Zhang (1993)). ...
... However, as the nomen for the genus Ermia was already in use for a genus of locusts and was preoccupied, Gumprecht and Tillack (2004) provided a replacement name for this taxon and the species was assigned to a monotypic genus Zhaoermia. The first molecular phylogenetic data on these snakes, provided by Guo et al. (2007), indicated close relationships between Zhaoermia and Triceratolepidophis and Protobothrops and both former genera were coined as subjective junior synonyms of Protobothrops (Guo et al. 2007). Protobothrops mangshanensis is one of the largest members of the genus and is clearly different from other congeners in both external morphology, colouration and skull morphology (see Zhang (1993)). ...
We describe a new species of lance-headed pit viper from north-western Laos, based on morphological and molecular (6092 bp from cyt b, ND4, COI, 12S rRNA and 16S rRNA mitochondrial DNA genes and c-mos and RAG1 nuclear DNA genes) lines of evidence. Protobothrops flavirostrissp. nov. is easily distinguished from its congeners by the following combination of morphological characters: dorsal scales in 23–21–17 rows, all keeled; ventral scales 215; subcaudal scales 79, all paired; supralabials 7–8; infralabials 10; horn-like projections on supraoculars absent; head triangular with a typical lance-shaped pattern on its dorsal surface; three faint dark vertical stripes on the snout; head blackish-brown with rostral, nasals, preoculars, loreals and the two anterior supralabials, as well as the anterior parts of supraoculars yellow-orange; dorsal surfaces of body and tail brown or greyish-brown, dorsum with large dark reddish-brown cross-shaped blotches, edged in black, somewhat fused together forming an interrupted zigzag line and a row of large brown ventrolateral blotches on each side. The new species differs from the morphologically similar species Protobothrops kelomohy by a significant divergence in cytochrome b mitochondrial DNA gene sequences (p = 7.8%). The new species is currently known only from tropical limestone forest of Vientiane Province, north-western Laos (elevation 362 m a.s.l.). We suggest the new species be considered as Endangered (EN) following the IUCN’s Red List categories.
... However, the effectiveness of antivenoms in neutralizing the venom of T. gracilis remains largely unexplored in specialized research studies. Although T. gracilis and T. stejnegeri were previously classified within the same genus, Trimeresurus, numerous recent studies on their phylogenetic relationships have indicated that they should not be grouped together [17][18][19][20]. Furthermore, recent research has revealed that the amino acid composition of some metalloproteinases, serine proteases, and phospholipase A 2 enzymes in the venom of T. gracilis is similar to those found in Asian pit vipers (Gloydius) and rattlesnakes (Crotalus) [21,22]. ...
Snakebite envenomation is a significant global health issue that requires specific antivenom treatments. In Taiwan, available antivenoms target a variety of snakes, but none specifically target Trimeresurus gracilis, an endemic and protected species found in the high mountain areas of Taiwan. This study evaluated the effectiveness of existing antivenoms against T. gracilis venom, focusing on a bivalent antivenom developed for Trimeresurus stejnegeri and Protobothrops mucrosquamatus (TsPmAV), as well as monovalent antivenoms for Deinagkistrodon acutus (DaAV) and Gloydius brevicaudus (GbAV). Our research involved in vivo toxicity testing in mice and in vitro immunobinding experiments using (chaotropic) enzyme-linked immunosorbent assays, comparing venoms from four pit viper species (T. gracilis, T. stejnegeri, P. mucrosquamatus, and D. acutus) with three types of antivenoms. These findings indicate that TsPmAV partially neutralized T. gracilis venom, marginally surpassing the efficacy of DaAV. In vitro tests revealed that GbAV displayed higher binding capacities toward T. gracilis venom than TsPmAV or DaAV. Comparisons of electrophoretic profiles also reveal that T. gracilis venom has fewer snake venom C-type lectin like proteins than D. acutus, and has more P-I snake venom metalloproteases or fewer phospholipase A2 than G. brevicaudus, T. stejnegeri, or P. mucrosquamatus. This study highlights the need for antivenoms that specifically target T. gracilis, as current treatments using TsPmAV show limited effectiveness in neutralizing local effects in patients. These findings provide crucial insights into clinical treatment protocols and contribute to the understanding of the evolutionary adaptation of snake venom, aiding in the development of more effective antivenoms for human health.
... Due to seven more members (P. dabieshanensis, P. himalayanus, P. kelomohy, P. mangshanensis, P. maolanensis, P. sieversorum, and P. trungkhanensis) having been designated or revised from synonymous species over the last two decades, habu snakes were recently recognized to comprise 15 validated species [16][17][18][19][20]. Phylogenetic analysis based on multilocus gene markers robustly classified 14 habu snakes into four clades, in which the first clade comprises P. himalayanus, P. kaulbacki, and P. sieversorum and the second clade only comprises P. mangshanensis, while the other two clades contain the remaining four and six Protobothrops species, respectively [21]. ...
We conducted a comparative analysis to unveil the divergence among venoms from a subset of Old World habu snakes (Protobothrops) in terms of venomic profiles and toxicological and enzymatic activities. A total of 14 protein families were identified in the venoms from these habu snakes, and 11 of them were shared among these venoms. The venoms of five adult habu snakes were overwhelmingly dominated by SVMP (32.56 ± 13.94%), PLA2 (22.93 ± 9.26%), and SVSP (16.27 ± 4.79%), with a total abundance of over 65%, while the subadult P. mangshanensis had an extremely low abundance of PLA2 (1.23%) but a high abundance of CTL (51.47%), followed by SVMP (22.06%) and SVSP (10.90%). Apparent interspecific variations in lethality and enzymatic activities were also explored in habu snake venoms, but no variations in myotoxicity were found. Except for SVSP, the resemblance of the relatives within Protobothrops in other venom traits was estimated to deviate from Brownian motion evolution based on phylogenetic signals. A comparative analysis further validated that the degree of covariation between phylogeny and venom variation is evolutionarily labile and varies among clades of closely related snakes. Our findings indicate a high level of interspecific variation in the venom proteomes of habu snakes, both in the presence or absence and the relative abundance of venom protein families, and that these venoms might have evolved under a combination of adaptive and neutral mechanisms.
... 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]. ...
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. ...
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. ...
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). ...
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.
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].
— 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.