Nepenthaceae is one of the largest carnivorous plant families and features ecological and morphological adaptations indicating an impressive adaptive radiation. However, investigation of evolutionary and taxonomic questions is hindered by poor phylogenetic understanding, with previous molecular studies based on limited loci and taxa. We use high-throughput sequencing with a target-capture methodology based on a 353-loci, probe set to recover sequences for 197 samples, representing 151 described or putative Nepenthes species. Phylogenetic analyses were performed using supermatrix and maximum quartet species tree approaches. Our analyses confirm five Western outlier taxa, followed by N. danseri, as successively sister to the remainder of the group. We also find mostly consistent recovery of two major Southeast Asian clades. The first contains common or widespread lowland species plus a Wallacean-New Guinean clade. Within the second clade, sects. Insignes and Tentaculatae are well supported, while geographically defined clades representing Sumatra, Indochina, Peninsular Malaysia, Palawan, Mindanao and Borneo are also consistently recovered. However, we find considerable conflicting signal at the site and locus level, and often unstable backbone relationships. A handful of Bornean taxa are inconsistently placed and require further investigation. We make further suggestions for a modified infra-generic classification of genus Nepenthes.
Family I.3 lipase is distinguished from other families by the amino acid sequence and secretion mechanism. Little is known about the evolutionary process driving these differences. This study attempt to understand how the diverse temperature stabilities of bacterial lipases from family I.3 evolved. To achieve that, eighty-three protein sequences sharing a minimum 30% sequence identity with Antarctic Pseudomonas sp. AMS8 lipase were used to infer phylogenetic tree. Using ancestral sequence reconstruction (ASR) technique, the last universal common ancestor (LUCA) sequence of family I.3 was reconstructed. A gene encoding LUCA was synthesized, cloned and expressed as inclusion bodies in E. coli system. Insoluble form of LUCA was refolded using urea dilution method and then purified using affinity chromatography. The purified LUCA exhibited an optimum temperature and pH at 70 ℃ and 10 respectively. Various metal ions increased or retained the activity of LUCA. LUCA also demonstrated tolerance towards various organic solvents in 25% v/v concentration. The finding from this study could support the understanding on temperature and environment during ancient time. In overall, reconstructed ancestral enzymes have improved physicochemical properties that make them suitable for industrial applications and ASR technique can be employed as a general technique for enzyme engineering.
We examined the phylogeny and biogeography of the glassperch family Ambassidae (Teleostei), which is widely distributed in the freshwater, brackish and marine coastal habitats across the Indo-West Pacific region. We first built a comprehensive time-calibrated phylogeny of Ambassidae using five genes. We then used this tree to reconstruct the evolution of the salinity preference and ancestral areas. Our results indicate that the two largest genera of Ambassidae, Ambassis and Parambassis, are each not monophyletic. The most recent common ancestor of Ambassidae was freshwater adapted and lived in Australia about 56 million years ago. Three independent freshwater-to-marine transitions are inferred, but no marine-to-freshwater ones. To explain the distribution of ambassids, we hypothesise two long-distance marine dispersal events from Australia. A first event was towards Southeast Asia during the early Cenozoic, followed by a second one towards Africa during mid-Cenozoic. The phylogenetic signal associated with the salinity adaptation of these events was not detected, possibly because of the selective extinction of intermediate marine lineages. The Ambassidae shares two characteristics with other freshwater fish groups distributed in continental regions surrounding the Indian Ocean: They are too young to support the hypothesis that their distribution is the result of the fragmentation of Gondwana, but they did not retain the phylogenetic signal of their marine dispersal.
The family Rhacophoridae is one of the most diverse amphibian families in Asia, for which taxonomic understanding is rapidly-expanding, with new species being described steadily, and at increasingly finer genetic resolution. Distance-based methods frequently have been used to justify or at least to bolster the recognition of new species, particularly in complexes of "cryptic" species where obvious morphological differentiation does not accompany speciation. However, there is no universally-accepted threshold to distinguish intra- from interspecific genetic divergence. Moreover, indiscriminant use of divergence thresholds to delimit species can result in over- or underestimation of species diversity. To explore the range of variation in application of divergence scales, and to provide a family-wide assessment of species-level diversity in Old-World treefrogs (family Rhacophoridae), we assembled the most comprehensive multi-locus phylogeny to date, including all 18 genera and approximately 247 described species (∼60% coverage). We then used the Automatic Barcode Gap Discovery (ABGD) method to obtain different species-delimitation schemes over a range of prior intraspecific divergence limits to assess the consistency of divergence thresholds used to demarcate current species boundaries. The species-rich phylogeny was able to identify a number of taxonomic errors, namely the incorrect generic placement of Chiromantis inexpectatus, which we now move to the genus Feihyla, and the specific identity of Rhacophorus bipunctatus from Peninsular Malaysia, which we tentatively reassign to R. rhodopus. The ABGD analysis demonstrated overlap between intra- and interspecific divergence limits: genetic thresholds used in some studies to synonymize taxa have frequently been used in other studies to justify the recognition of new species. This analysis also highlighted numerous groups that could potentially be split or lumped, which we earmark for future examination. Our large-scale and en bloc approach to species-level phylogenetic systematics contributes to the resolution of taxonomic uncertainties, reveals possible new species, and identifies numerous groups that require critical examination. Overall, we demonstrate that the taxonomy and evolutionary history of Old-World tree frogs are far from resolved, stable or adequately characterized at the level of genus, species, and/or population.
Phylogeography can provide insight into the potential for speciation and identify geographic regions and evolutionary processes associated with species richness and evolutionary endemism. In the marine environment, highly mobile species sometimes show structured patterns of diversity, but the processes isolating populations and promoting differentiation are often unclear. The Delphinidae (oceanic dolphins) are a striking case in point and, in particular, bottlenose dolphins (Tursiops spp.). Understanding the radiation of species in this genus is likely to provide broader inference about the processes that determine patterns of biogeography and speciation, because both fine-scale structure over a range of kilometers and relative panmixia over an oceanic range are known for Tursiops populations. In our study, novel Tursiops spp. sequences from the northwest Indian Ocean (including mitogenomes and two nuDNA loci) are included in a worldwide Tursiops spp. phylogeographic analysis. We discover a new 'aduncus' type lineage in the Arabian Sea (off India, Pakistan and Oman) that diverged from the Australasian lineage ∼261 Ka. Effective management of coastal dolphins in the region will need to consider this new lineage as an evolutionarily significant unit. We propose that the establishment of this lineage could have been in response to climate change during the Pleistocene and show data supporting hypotheses for multiple divergence events, including vicariance across the Indo-Pacific barrier and in the northwest Indian Ocean. These data provide valuable transferable inference on the potential mechanisms for population and species differentiation across this geographic range.
Mitochondrial genomes provided the first widely used sequences that were sufficiently informative to resolve relationships among animals across a wide taxonomic domain, from within species to between phyla. However, mitogenome studies supported several anomalous relationships and fell partly out of favour as sequencing multiple, independent nuclear loci proved to be highly effective. A tendency to blame mitochondrial DNA (mtDNA) has overshadowed efforts to understand and ameliorate underlying model misspecification. Here we find that influential assessments of the infidelity of mitogenome phylogenies have often been overstated, but nevertheless, substitution saturation and compositional non-stationarity substantially mislead reconstruction. We show that RY coding the mtDNA, excluding protein-coding 3rd codon sites, partitioning models based on amino acid hydrophobicity and enhanced taxon sampling improve the accuracy of mitogenomic phylogeny reconstruction for placental mammals, almost to the level of multi-gene nuclear datasets. Indeed, combined analysis of mtDNA with 3-fold longer nuclear sequence data either maintained or improved upon the nuclear support for all generally accepted clades, even those that mtDNA alone did not favour, thus indicating "hidden support". Confident mtDNA phylogeny reconstruction is especially important for understanding the evolutionary dynamics of mitochondria themselves, and for merging extinct taxa into the tree of life, with ancient DNA often only accessible as mtDNA. Our ancient mtDNA analyses lend confidence to the relationships of three extinct megafaunal taxa: glyptodonts are nested within armadillos, the South American ungulate, Macrauchenia is sister to horses and rhinoceroses, and sabre-toothed and scimitar cats are the monophyletic sister-group of modern cats.
The mountains of Borneo are well known for their high endemicity and historical role in preserving Southeast Asian rainforest biodiversity, but the diversification of populations inhabiting these mountains is poorly studied. Here we examine the genetic structure of 12 Bornean montane passerines by comparing complete mtDNA ND2 gene sequences of populations spanning the island. Maximum likelihood and Bayesian phylogenetic trees and haplotype networks are examined for common patterns that might signal important historical events or boundaries to dispersal. Morphological and ecological characteristics of each species are also examined using phylogenetic generalized least-squares (PGLS) for correlation with population structure. Populations in only four of the 12 species are subdivided into distinct clades or haplotype groups. Although this subdivision occurred at about the same time in each species (ca. 0.6-0.7Ma), the spatial positioning of the genetic break differs among the species. In two species, northeastern populations are genetically divergent from populations elsewhere on the island. In the other two species, populations in the main Bornean mountain chain, including the northeast, are distinct from those on two isolated peaks in northwestern Borneo. We suggest different historical forces played a role in shaping these two distributions, despite commonality in timing. PGLS analysis showed that only a single characteristic-hand-wing index-is correlated with population structure. Birds with longer wings, and hence potentially more dispersal power, have less population structure. To understand historical forces influencing montane population structure on Borneo, future studies must compare populations across the entirety of Sundaland.
In order to clarify the phylogenetic relationships among the main marine myxosporean clades including newly established Ceratonova clade and scrutinizing their evolutionary origins, we performed large-scale phylogenetic analysis of all myxosporean species from the marine myxosporean lineage based on three gene analyses and statistical topology tests. Furthermore, we obtained new molecular data for Ceratonova shasta, C. gasterostea, eight Ceratomyxa species and one Myxodavisia species. We described five new species: Ceratomyxa ayami n. sp., C. leatherjacketi n. sp., C. synaphobranchi n. sp., C. verudaensis n. sp. and Myxodavisia bulani n. sp.; two of these formed a new, basal Ceratomyxa subclade. We identified that the Ceratomyxa clade is basal to all other marine myxosporean lineages, and Kudoa with Enteromyxum are the most recently branching clades. Topologies were least stable at the nodes connecting the marine urinary clade, the marine gall bladder clade and the Ceratonova clade. Bayesian inference analysis of SSU rDNA and the statistical tree topology tests suggested that Ceratonova is closely related to the Enteromyxum and Kudoa clades, which represent a large group of histozoic species. A close relationship between Ceratomyxa and Ceratonova was not supported, despite their similar myxospore morphologies. Overall, the site of sporulation in the vertebrate host is a more accurate predictor of phylogenetic relationships than the morphology of the myxospore.
Two Unionida (freshwater mussel) families are present in the Northern Hemisphere; the Margaritiferidae, representing the most threatened of unionid families, and the Unionidae, which include several genera of unresolved taxonomic placement. The recent reassignment of the poorly studied Lamprotula rochechouartii from the Unionidae to the Margaritiferidae motivated a new search for other potential species of margaritiferids from members of Gibbosula and Lamprotula. Based on molecular and morphological analyses conducted on newly collected specimens from Vietnam, we here assign Gibbosula crassa to the Margaritiferidae. Additionally, we reanalyzed all diagnostic characteristics of the Margaritiferidae and examined museum specimens of Lamprotula and Gibbosula. As a result, two additional species are also moved to the Margaritiferidae, i.e. Gibbosula confragosa and Gibbosula polysticta. We performed a robust five marker phylogeny with all available margaritiferid species and discuss the taxonomy within the family. The present phylogeny reveals the division of Margaritiferidae into four ancient clades with distinct morphological, biogeographical and ecological characteristics that justify the division of the Margaritiferidae into two subfamilies (Gibbosulinae and Margaritiferinae) and four genera (Gibbosula, Cumberlandia, Margaritifera, and Pseudunio). The systematics of the Margaritiferidae family is re-defined as well as their distribution, potential origin and main biogeographic patterns.
Wolbachia pipientis is a diverse, ubiquitous and most prevalent intracellular bacterial group of alpha-Proteobacteria that is concerned with many biological processes in arthropods. The coconut hispine beetle (CHB), Brontispa longissima (Gestro) is an economically important pest of palm cultivation worldwide. In the present study, we comprehensively surveyed the Wolbachia-infection prevalence and mitochondrial DNA (mtDNA) polymorphism in CHB from five different geographical locations, including China's Mainland and Taiwan, Vietnam, Thailand, Malaysia and Indonesia. A total of 540 sequences were screened in this study through three different genes, i.e., cytochrome oxidase subunit I (COI), Wolbachia outer surface protein (wsp) and multilocus sequencing type (MLST) genes. The COI genetic divergence ranges from 0.08% to 0.67%, and likewise, a significant genetic diversity (π = 0.00082; P = 0.049) was noted within and between all analyzed samples. In the meantime, ten different haplotypes (H) were characterized (haplotype diversity = 0.4379) from 21 different locations, and among them, H6 (46 individuals) have shown a maximum number of population clusters than others. Subsequently, Wolbachia-prevalence results indicated that all tested specimens of CHB were found positive (100%), which suggested that CHB was naturally infected with Wolbachia. Wolbachia sequence results (wsp gene) revealed a high level of nucleotide diversity (π = 0.00047) under Tajima's D test (P = 0.049). Meanwhile, the same trend of nucleotide diversity (π = 0.00041) was observed in Wolbachia concatenated MLST locus. Furthermore, phylogenetic analysis (wsp and concatenated MLST genes) revealed that all collected samples of CHB attributed to same Wolbachia B-supergroup. Our results strongly suggest that Wolbachia bacteria and mtDNA were highly concordant with each other and Wolbachia can affect the genetic structure and diversity within the CHB populations.
Recent morphological and molecular studies led to the recognition of two extant species of clouded leopards; Neofelis nebulosa from mainland southeast Asia and Neofelis diardi from the Sunda Islands of Borneo and Sumatra, including the Batu Islands. In addition to these new species-level distinctions, preliminary molecular data suggested a genetic substructure that separates Bornean and Sumatran clouded leopards, indicating the possibility of two subspecies of N. diardi. This suggestion was based on an analysis of only three Sumatran and seven Bornean individuals. Accordingly, in this study we re-evaluated this proposed subspecies differentiation using additional molecular (mainly historical) samples of eight Bornean and 13 Sumatran clouded leopards; a craniometric analysis of 28 specimens; and examination of pelage morphology of 20 museum specimens and of photographs of 12 wild camera-trapped animals. Molecular (mtDNA and microsatellite loci), craniomandibular and dental analyses strongly support the differentiation of Bornean and Sumatran clouded leopards, but pelage characteristics fail to separate them completely, most probably owing to small sample sizes, but it may also reflect habitat similarities between the two islands and their recent divergence. However, some provisional discriminating pelage characters are presented that need further testing. According to our estimates both populations diverged from each other during the Middle to Late Pleistocene (between 400 and 120 kyr). We present a discussion on the evolutionary history of Neofelis diardi sspp. on the Sunda Shelf, a revised taxonomy for the Sunda clouded leopard, N. diardi, and formally describe the Bornean subspecies, Neofelis diardi borneensis, including the designation of a holotype (BM.3.4.9.2 from Baram, Sarawak) in accordance with the rules of the International Code of Zoological Nomenclature.
Many marine species are specialized to specific parts of a habitat. In a mangrove forest, for instance, species may be restricted to the mud surface, the roots and trunks of mangrove trees, or rotting logs, which can be regarded as distinct microhabitats. Shifts to new microhabitats may be an important driver of sympatric speciation. However, the evolutionary history of these shifts is still poorly understood in most groups of marine organisms, because it requires a well-supported phylogeny with relatively complete taxon sampling. Onchidiid slugs are an ideal case study for the evolutionary history of habitat and microhabitat shifts because onchidiid species are specialized to different tidal zones and microhabitats in mangrove forests and rocky shores, and the taxonomy of the family in the Indo-West Pacific has been recently revised in a series of monographs. Here, DNA sequences for onchidiid species from the North and East Pacific, the Caribbean, and the Atlantic are used to reconstruct phylogenetic relationships among Onchidella species, and are combined with new data for Indo-West Pacific species to reconstruct a global phylogeny of the family. The phylogenetic relationships of onchidiid slugs are reconstructed based on three mitochondrial markers (COI, 12S, 16S) and three nuclear markers (28S, ITS2, H3) and nearly complete taxon sampling (all 13 genera and 62 of the 67 species). The highly-supported phylogeny presented here suggests that ancestral onchidiids most likely lived in the rocky intertidal, and that a lineage restricted to the tropical Indo-West Pacific colonized new habitats, including mudflats, mangrove forests, and high-elevation rainforests. Many onchidiid species in the Indo-West Pacific diverged during the Miocene, around the same time that a high diversity of mangrove plants appears in the fossil record, while divergence among Onchidella species occurred earlier, likely beginning in the Eocene. It is demonstrated that ecological specialization to microhabitats underlies the divergence between onchidiid genera, as well as the diversification through sympatric speciation in the genera Wallaconchis and Platevindex. The geographic distributions of onchidiid species also indicate that allopatric speciation played a key role in the diversification of several genera, especially Onchidella and Peronia. The evolutionary history of several morphological traits (penial gland, rectal gland, dorsal eyes, intestinal loops) is examined in relation to habitat and microhabitat evolutionary transitions and suggests that the rectal gland of onchidiids is an adaptation to high intertidal and terrestrial habitats.
In the species groups related to Diphasiastrum multispicatum and D. veitchii, hybridization was investigated in samples from northern and southern Vietnam and the island of Taiwan, including available herbarium specimens from southeast Asia. The accessions were analyzed using flow cytometry (living material only), Sanger sequencing and multiplexed inter-simple sequence repeat genotyping by sequencing. We detected two cases of ancient hybridization involving different combinations of parental species; both led via subsequent duplication to tetraploid taxa. A cross D. multispicatum × D. veitchii from Malaysia represents D. wightianum, a tetraploid taxon according to reported DNA content measurements of dried material (genome formulas MM, VV and MMVV, respectively). The second case involves D. veitchii and an unknown diploid parent (genome formula XX). Three hybridogenous taxa (genome formulas VVX, VVXX, VVVX) were discernable by a combination of flow cytometry and molecular data. Taxon I (VVX, three clones found on Taiwan island) is apparently triploid. Taxon II represents another genetically diverse and sexual tetraploid species (VVXX) and can be assigned to D. yueshanense, described from Taiwan island but occurring as well in mainland China and Vietnam. Taxon III is as well most likely tetraploid (VVVX) and represented by at least one, more likely two, clones from Taiwan island. Taxa I and III are presumably asexual and new to science. Two independently inherited nuclear markers recombine only within, not between these hybrids, pointing towards reproductive isolation. We present an evolutionary scheme which explains the origin of the hybrids and the evolution of new and fully sexual species by hybridization and subsequent allopolyploidization in flat-branched clubmosses.
In widespread species, the diverse ecological conditions in which the populations occur, and the presence of many potential geographical barriers through their range are expected to have created ample opportunities for the evolution of distinct, often cryptic lineages. In this work, we tested for species boundaries in one such widespread species, the king cobra, Ophiophagus hannah (Cantor, 1836), a largely tropical elapid snake distributed across the Oriental realm. Based on extensive geographical sampling across most of the range of the species, we initially tested for candidate species (CS) using Maximum-Likelihood analysis of mitochondrial genes. We then tested the resulting CS using both morphological data and sequences of three single-copy nuclear genes. We used snapclust to determine the optimal number of clusters in the nuclear dataset, and Bayesian Phylogenetics and Phylogeography (BPP) to test for likely species status. We used non-metric multidimensional scaling (nMDS) analysis for discerning morphological separation. We recovered four independently evolving, geographically separated lineages that we consider Confirmed Candidate Species: (1) Western Ghats lineage; (2) Indo-Chinese lineage (3) Indo-Malayan lineage; (4) Luzon Island lineage, in the Philippine Archipelago. We discuss patterns of lineage divergence, particularly in the context of low morphological divergence, and the conservation implications of recognizing several endemic king cobra lineages.
The increased rate at which complete mitogenomes are being sequenced and their increasing use for phylogenetic studies have resulted in a bioinformatic bottleneck in preparing and utilising such data for phylogenetic analysis. Hence, we present MitoPhAST, an automated tool that (1) identifies annotated protein-coding gene features and generates a standardised, concatenated and partitioned amino acid alignment directly from complete/partial GenBank/EMBL-format mitogenome flat files, (2) generates a maximum likelihood phylogenetic tree using optimised protein models and (3) reports various mitochondrial genes and sequence information in a table format. To demonstrate the capacity of MitoPhAST in handling a large dataset, we used 81 publicly available decapod mitogenomes, together with eight new complete mitogenomes of Australian freshwater crayfishes, including the first for the genus Gramastacus, to undertake an updated test of the monophyly of the major groups of the order Decapoda and their phylogenetic relationships. The recovered phylogenetic trees using both Bayesian and ML methods support the results of studies using fragments of mtDNA and nuclear markers and other smaller-scale studies using whole mitogenomes. In comparison to the fragment-based phylogenies, nodal support values are generally higher despite reduced taxon sampling suggesting there is value in utilising more fully mitogenomic data. Additionally, the simple table output from MitoPhAST provides an efficient summary and statistical overview of the mitogenomes under study at the gene level, allowing the identification of missing or duplicated genes and gene rearrangements. The finding of new mtDNA gene rearrangements in several genera of Australian freshwater crayfishes indicates that this group has undergone an unusually high rate of evolutionary change for this organelle compared to other major families of decapod crustaceans. As a result, freshwater crayfishes are likely to be a useful model for studies designed to understand the evolution of mtDNA rearrangements. We anticipate that our bioinformatics pipeline will substantially help mitogenome-based studies increase the speed, accuracy and efficiency of phylogenetic studies utilising mitogenome information. MitoPhAST is available for download at https://github.com/mht85/MitoPhAST.
Amphiboloidea is a small but widespread group of snails found exclusively, and often abundantly, in mudflat and associated salt marsh or mangrove habitat. This study uses molecular data from three loci (COI, 16S and 28S) to infer phylogenetic relationships in Amphiboloidea and examine its position in Euthyneura. All but two of the named extant species of Amphiboloidea and additional undescribed taxa from across Southeast Asia and the Arabian Gulf were sampled. In contrast to the current morphology-based classification dividing Amphiboloidea into three families, analysis of molecular data supports revision of the classification to comprise two families. Maningrididae is a monotypic family basal to Amphibolidae, which is revised to comprise three subfamilies: Amphibolinae, Phallomedusinae and Salinatorinae. Sequence divergence between Asian populations of Naranjia is relatively large and possibly indicative of species complexes divergent across the Strait of Malacca. Salinatorrosacea and Salinator burmana do not cluster with other Salinator species, and require generic reassignment. In addition, sequences were obtained from an undescribed species of Lactiforis from the Malay Peninsula. Reconstruction of ancestral distributions indicates a plesiomorphic distribution and centre of origin in Australasia, with two genera subsequently diversifying throughout Asia. Increasing the sampling density of amphiboloid taxa in a phylogenetic analysis of Euthyneura did not resolve the identity of the sister taxon to Amphibolidae, but confirmed its inclusion in Pulmonata/Panpulmonata.
Platanthera is one of the largest genera of temperate orchids in the Holarctic and exemplifies a lineage that has adaptively radiated into diverse habitats within North America, Asia, Europe, North Africa, Borneo, and Sarawak. Major centers of diversity in this genus are North America and eastern Asia. Despite its diversity, a thorough phylogenetic hypothesis for the genus is lacking because no studies have yet sampled taxa exhaustively or developed a robust molecular toolkit. While there is strong evidence that suggests monophyly of subgenus Limnorchis, most taxa in this group have not been included in a phylogenetic analysis. In this study, we developed a new toolkit for Platanthera consisting of genomic information from 617 low-copy nuclear loci. Using a targeted enrichment approach, we collected high-throughput sequence data in 23 accessions of nine of the 12 diploid species of subgenus Limnorchis and outgroup species across Platanthera. A maximum likelihood analysis resolved a strongly supported monophyletic clade for subgenus Limnorchis. Ancestral biogeographic reconstruction indicated that subgenus Limnorchis originated in western North America ca. 3-4.5 Mya from an ancestor that was widespread in western North America and eastern Asia and subsequently diversified in western North America, followed by dispersal of some species to eastern North America. Our results indicate complex biogeographic connections between Asia and North America, and therefore it suggests that Platanthera is a suitable system to test biogeographic hypotheses over time and space in the Holarctic. Our results are also expected to facilitate further study of diversification and biogeographic spread across Platanthera and lay the groundwork for understanding independent origins, biogeography, and morphological diversification of polyploid species within subgenus Limnorchis.