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.
A fanged frog Limnonectes kuhlii was once thought to be wide-ranging in Southeast Asia, but is now confined to its type locality Java through recent phylogenetic studies, which clarified heterospecific status of non-Javanese populations, and monophyly of Bornean populations. However, large genetic differences among Bornean populations suggest occurrence of cryptic species, which we test using dense geographic sampling. We estimated the phylogenetic relationships among samples of Bornean populations together with their putative relatives from the continental Southeast Asia, using 2517bp sequences of the 12S rRNA, tRNA(val), and 16S rRNA of mitochondrial DNA, and 2367bp sequences of the NCX1, POMC, and RAG1 of nuclear genes. In the mtDNA trees, Bornean L. kuhlii-like frogs formed a monophyletic group split into 18 species lineages including L. hikidai, with the deepest phylogenetic split separating L. cintalubang from the remaining species. Almost all of these lineages co-occur geographically, and two to three lineages were found syntopically in each locality. Co-occurrence of more than one lineage may be maintained by differential morphology and microhabitat selection. These syntopic lineages should be regarded as distinct species. Our results clearly indicate that taxonomic revision is urgent to clarify many evolutionary problems of Bornean L. kuhlii-like frogs.
Freshwater mussels of the order Unionida are key elements of freshwater habitats and are responsible for important ecological functions and services. Unfortunately, these bivalves are among the most threatened freshwater taxa in the world. However, conservation planning and management are hindered by taxonomic problems and a lack of detailed ecological data. This highlights the urgent need for advances in the areas of systematics and evolutionary relationships within the Unionida. This study presents the most comprehensive phylogeny to date of the larger Unionida family, i.e., the Unionidae. The phylogeny is based on a combined dataset of 1032bp (COI+28S) of 70 species in 46 genera, with 7 of this genera being sequenced for the first time. The resulting phylogeny divided the Unionidae into 6 supported subfamilies and 18 tribes, three of which are here named for the first time (i.e., Chamberlainiini nomen novum, Cristariini nomen novum and Lanceolariini nomen novum). Molecular analyses were complemented by investigations of selected morphological, anatomical and behavioral characters used in traditional phylogenetic studies. No single morphological, anatomical or behavioral character was diagnostic at the subfamily level and few were useful at the tribe level. However, within subfamilies, many tribes can be recognized based on a subset of these characters. The geographical distribution of each of the subfamilies and tribes is also presented. The present study provides important advances in the systematics of these extraordinary taxa with implications for future ecological and conservation studies.
Frullania subgenus Microfrullania is a clade of ca. 15 liverwort species occurring in Australasia, Malesia, and southern South America. We used combined nuclear and chloroplast sequence data from 265 ingroup accessions to test species circumscriptions and estimate the biogeographic history of the subgenus. With dense infra-specific sampling, we document an important role of long-distance dispersal in establishing phylogeographic patterns of extant species. At deeper time scales, a combination of phylogenetic analyses, divergence time estimation and ancestral range estimation were used to reject vicariance and to document the role of long-distance dispersal in explaining the evolution and biogeography of the clade across the southern Hemisphere. A backbone phylogeny for the subgenus is proposed, providing insight into evolution of morphological patterns and establishing the basis for an improved sectional classification of species within Microfrullania. Several species complexes are identified, the presence of two undescribed but genetically and morphologically distinct species is noted, and previously neglected names are discussed.
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.
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.
The infraorder Anomura consists of a morphologically and ecologically heterogeneous group of decapod crustaceans, and has attracted interest from taxonomists for decades attempting to find some order out of the seemingly chaotic diversity within the group. Species-level diversity within the Anomura runs the gamut from the "hairy" spindly-legged yeti crab found in deep-sea hydrothermal vent environments to the largest known terrestrial invertebrate, the robust coconut or robber crab. Owing to a well-developed capacity for parallel evolution, as evidenced by the occurrence of multiple independent carcinization events, Anomura has long tested the patience and skill of both taxonomists attempting to find order, and phylogeneticists trying to establish stable hypotheses of evolutionary inter-relationships. In this study, we performed genome skimming to recover the mitogenome sequences of 12 anomuran species including the world's largest extant invertebrate, the robber crab (Birgus latro), thereby over doubling these resources for this group, together with 8 new brachyuran mitogenomes. Maximum-likelihood (ML) and Bayesian-inferred (BI) phylogenetic reconstructions based on amino acid sequences from mitogenome protein-coding genes provided strong support for the monophyly of the Anomura and Brachyura and their sister relationship, consistent with previous studies. The majority of relationships within families were supported and were largely consistent with current taxonomic classifications, whereas many relationships at higher taxonomic levels were unresolved. Nevertheless, we have strong support for a polyphyletic Paguroidea and recovered a well-supported clade of a subset of paguroids (Diogenidae + Coenobitidae) basal to all other anomurans, though this requires further testing with greater taxonomic sampling. We also introduce a new feature to the MitoPhAST bioinformatics pipeline (https://github.com/mht85/MitoPhAST) that enables the extraction of mitochondrial gene order (MGO) information directly from GenBank files and clusters groups based on common MGOs. Using this tool, we compared MGOs across the Anomura and Brachyura, identifying Anomura as a taxonomic "hot spot" with high variability in MGOs among congeneric species from multiple families while noting the broad association of highly-rearranged MGOs with several anomuran lineages inhabiting extreme niches. We also demonstrate the value of MGOs as a source of novel synapomorphies for independently reinforcing tree-based relationships and for shedding light on relationships among challenging groups such as the Aegloidea and Lomisoidea that were unresolved in phylogenetic reconstructions. Overall, this study contributes a substantial amount of new genetic material for Anomura and attempts to further resolve anomuran evolutionary relationships where possible based on a combination of sequence and MGO information. The new feature in MitoPhAST adds to the relatively limited number of bioinformatics tools available for MGO analyses, which can be utilized widely across animal groups.
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.
Myrteae (c. 2500 species; 51 genera) is the largest tribe of Myrtaceae and an ecologically important groups of angiosperms in the Neotropics. Systematic relationships in Myrteae are complex, hindering conservation initiatives and jeopardizing evolutionary modelling. A well-supported and robust phylogenetic hypothesis was here targeted towards a comprehensive understanding of the relationships within the tribe. The resultant topology was used as a base for key evolutionary analyses such as age estimation, historical biogeography and diversification rate patterns. One nuclear (ITS) and seven chloroplast (psbA-trnH, matK, ndhF, trnl-trnF, trnQ-rps16, rpl16 and rpl32-trnL) DNA regions for 115 taxa representing 46 out of the 51 genera in the tribe were accessed and analysed using maximum likelihood and Bayesian inference tools for phylogenetic reconstruction. Dates of diversification events were estimated and contrasted using two distinct fossil sets (macro and pollen) in BEAST. The subsequent dated phylogenies were compared and analysed for biogeographical patterns using BioGeoBEARS and diversification rates using BAMM. Myrteae phylogeny presents strong statistical support for three major clades within the tribe: Australasian group, Myrtus group and Main Neotropical Lineage. Dating results from calibration using macrofossil are an average of 20 million years older and show an early Paleocene origin of Myrteae, against a mid-Eocene one from the pollen fossil calibration. Biogeographic analysis shows the origin of Myrteae in Zealandia in both calibration approaches, followed by a widespread distribution throughout the still-linked Gondwana continents and diversification of Neotropical endemic lineages by later vicariance. Best configuration shift indicates three points of acceleration in diversification rates, all of them occurring in the Main Neotropical Lineage. Based on the reconstructed topology, several new taxonomic placements were recovered, including: the relative position of Myrtus communis, the placement of the Blepharocalyx group, the absence of generic endemism in the Caribbean, and the paraphyletism of the former Pimenta group. Distinct calibration approaches affect biogeography interpretation, increasing the number of necessary long distance dispersal events in the topology with older nodes. It is hypothesised that biological intrinsic factors such as modifications of embryo type and polyploidy might have played a role in accelerating shifts of diversification rates in Neotropical lineages. Future perspectives include formal subtribal classification, standardization of fossil calibration approaches and better links between diversification shifts and trait evolution.
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.
The gekkonid genus Cyrtodactylus is a highly diverse group of lizards (280 + species), which covers an expansive geographic range. Although this genus has been the focus of many taxonomic and molecular systematic studies, species on the Southeast Asian island of Borneo have remained understudied, leading to an unclear evolutionary history with cascading effects on taxonomy and biogeographic inferences. We assembled the most comprehensive multilocus Bornean dataset (one mitochondrial and three nuclear loci) that included 129 novel sequences and representatives from each known Cyrtodactylus species on the island to validate taxonomic status, assess species diversity, and elucidate biogeographic patterns. Our results uncovered a high proportion of cryptic diversity and revealed numerous taxonomic complications, especially within the C. consobrinus, C. malayanus, and C. pubisulcus groups. Comparisons of pairwise genetic distances and a preliminary species delimitation analysis using the Automatic Barcode Gap Discovery (ABGD) method demonstrated that some wide-ranging species on Borneo likely comprise multiple distinct and deeply divergent lineages, each with more restricted distributional ranges. We also tested the prevailing biogeographic hypothesis of a single invasion from Borneo into the Philippines. Our analyses revealed that Philippine taxa were not monophyletic, but were likely derived from multiple separate invasions into the geopolitical areas comprising the Philippines. Although our investigation of Bornean Cyrtodactylus is the most comprehensive to-date, it highlights the need for expanded taxonomic sampling and suggests that our knowledge of the evolutionary history, systematics, and biogeography of Bornean Cyrtodactylus is far from complete.
Western Southeast Asia is hosting one of the world's most diverse faunas, and one of the reasons for this huge diversity is the complex geologic past of the area, increasing the frequency of isolation and expansion events over evolutionary time scale. As an example case, the present study reveals the phylogeny and biogeographic history of the Paracanthocobitis zonalternans species complex, small benthic freshwater fish (Teleostei: Nemacheilidae) that are commonly occurring across western Southeast Asia (from central Myanmar through western and southern Thailand to northern Malaysia). The group is particularly interesting since it occurs in three biogeographic subdivisions (Indian, Indochinese, Malay/Sundaic) and across all of the major biogeographic barriers in the region. Basing on mitochondrial and nuclear sequence data of 93 samples from about 50 localities we found six major clades, most with exclusive geographic distribution. Divergence time dated the origin of the P. zonalternans species complex to early Miocene (17.8 MYA) and a biogeographic analysis identified the Tenasserim region as the ancestral region. From this region the fish spread during periods of lowered global sea level, particularly during late Miocene (11-8 MYA) northwards into all Burmese river basins and southwards into south Thailand and northern Malaysia. Besides lowered global sea level periods, local stream capture events allowed the complex to expand, e.g. into the Mae Klong basin. Strong fragmentations during periods with elevated sea level during the Pliocene and Pleistocene repeatedly restricted populations to refuges and shaped the observed major lineages. Our results document a higher diversity within the P. zonalternans species complex than formerly believed and a strong impact of global sea level on its evolutionary history. Low sea levels promoted dispersal and elevated sea levels fragmentation events. A very similar impact of sea level changes can be expected in all stationary fauna (freshwater and terrestrial) in all non-mountainous coastal regions worldwide.
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.
One of the most urgent contemporary tasks for taxonomists and evolutionary biologists is to estimate the number of species on earth. Recording alpha diversity is crucial for protecting biodiversity, especially in areas of elevated species richness, which coincide geographically with increased anthropogenic environmental pressures - the world's so-called biodiversity hotspots. Although the distribution of Puddle frogs of the genus Occidozyga in South and Southeast Asia includes five biodiversity hotspots, the available data on phylogeny, species diversity, and biogeography are surprisingly patchy. Samples analyzed in this study were collected throughout Southeast Asia, with a primary focus on Sundaland and the Philippines. A mitochondrial gene region comprising ~ 2000 bp of 12S and 16S rRNA with intervening tRNA Valine and three nuclear loci (BDNF, NTF3, POMC) were analyzed to obtain a robust, time-calibrated phylogenetic hypothesis. We found a surprisingly high level of genetic diversity within Occidozyga, based on uncorrected p-distance values corroborated by species delimitation analyses. This extensive genetic diversity revealed 29 evolutionary lineages, defined by the > 5% uncorrected p-distance criterion for the 16S rRNA gene, suggesting that species diversity in this clade of phenotypically homogeneous forms probably has been underestimated. The comparison with results of other anuran groups leads to the assumption that anuran species diversity could still be substantially underestimated in Southeast Asia in general. Many genetically divergent lineages of frogs are phenotypically similar, indicating a tendency towards extensive morphological conservatism. We present a biogeographic reconstruction of the colonization of Sundaland and nearby islands which, together with our temporal framework, suggests that lineage diversification centered on the landmasses of the northern Sunda Shelf. This remarkably genetically structured group of amphibians could represent an exceptional case for future studies of geographical structure and diversification in a widespread anuran clade spanning some of the most pronounced geographical barriers on the planet (e.g., Wallace's Line). Studies considering gene flow, morphology, ecological and bioacoustic data are needed to answer these questions and to test whether observed diversity of Puddle frog lineages warrants taxonomic recognition.
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.
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.
The Arracacia clade (Apiaceae, Apioideae) is a heterogeneous assemblage of 12 genera, comprising 111 known species distributed in high montane temperate and sub-alpine habitats of meso- and South America. Previous studies have indicated that the genera Arracacia, Coulterophytum, and Prionosciadium are polyphyletic, but for the most part relationships among the members of the clade are largely unknown. Initially, cladistic analyses of nrDNA ITS sequences were carried out on 212 accessions (122 taxa), representing 92 species of the Arracacia clade and outgroups from the closely-related páramo genera Cotopaxia, Niphogeton, and Perissocoeleum and members of the Perennial Endemic North American clade and its allies. Using the ITS results to inform sampling of a small subset of taxa, a pilot study examining the phylogenetic utility of 20 noncoding chloroplast loci was subsequently performed to identify those regions most useful at resolving relationships. A cost-benefit analysis determined that five loci (trnQ-5'rps16, trnD-trnT, rpl32-trnL, psbD-trnT, ndhA intron) would maximize resolution and branch support in the clade. Cladistic analyses of four of these loci (trnQ-5'rps16, trnD-trnT, rpl32-trnL, ndhA intron) and the ITS region, separately and combined, revealed that Arracacia, Coaxana, Coulterophytum, Prionosciadium, and Rhodosciadium are each polyphyletic and that Donnellsmithia and Myrrhidendron are each monophyletic. Although most relationships in the Arracacia clade and among the closely-related genera Cotopaxia, Niphogeton, and Perissocoeleum are poorly resolved and supported, ten groups are recognized for future revisionary studies. Polyploidy and rapid species radiation have likely confounded generic circumscriptions and interpretation of relationships.
To further understand the evolutionary history and mitogenomic features of Australia's highly distinctive freshwater crayfish fauna, we utilized a recently described rapid mitogenome sequencing pipeline to generate 24 new crayfish mitogenomes including a diversity of burrowing crayfish species and the first for Astacopsis gouldi, the world's largest freshwater invertebrate. Whole mitogenome-based phylogeny estimates using both Bayesian and Maximum Likelihood methods substantially strengthen existing hypotheses for systematic relationships among Australian freshwater crayfish with evidence of pervasive diversifying selection and accelerated mitochondrial substitution rate among the members of the clade representing strongly burrowing crayfish that may reflect selection pressures for increased energy requirement for adaptation to terrestrial environment and a burrowing lifestyle. Further, gene rearrangements are prevalent in the burrowing crayfish mitogenomes involving both tRNA and protein coding genes. In addition, duplicated control regions were observed in two closely related Engaeus species, together with evidence for concerted evolution. This study significantly adds to the understanding of Australian freshwater crayfish evolutionary relationships and suggests a link between mitogenome evolution and adaptation to terrestrial environments and a burrowing lifestyle in freshwater crayfish.
As opposed to angiosperms, moss species richness is similar among tropical regions of the world, in line with the hypothesis that tropical bryophytes are extremely good dispersers. Here, we reconstructed the phylogeny of the pantropical moss genus Pelekium to test the hypothesis that high migration rates erase any difference in species richness among tropical regions. In contrast with this hypothesis, several species considered to have a pantropical range were resolved as a complex of species with a strong geographic structure. Consequently, a significant phylogeographical signal was found in the data, evidencing that cladogenetic diversification within regions takes place at a faster rate than intercontinental migration. The shape of the Pelekium phylogeny, along with the selection of a constant-rate model of diversification among species in the genus, suggests, however, that the cladogenetic speciation patterns observed in Pelekium are not comparable to some of the spectacular examples of tropical radiations reported in angiosperms. Rather, the results presented here point to the constant accumulation of diversity through time in Pelekium. This, combined with evidence for long-distance dispersal limitations in the genus, suggests that the similar patterns of species richness among tropical areas are better explained in terms of comparable rates of diversification across tropical regions than by the homogenization of species richness by recurrent migrations.
Inferring interfamilial relationships within the eudicot order Ericales has remained one of the more recalcitrant problems in angiosperm phylogenetics, likely due to a rapid, ancient radiation. As a result, no comprehensive time-calibrated tree or biogeographical analysis of the order has been published. Here, we elucidate phylogenetic relationships within the order and then conduct time-dependent biogeographical and diversification analyses by using a taxon and locus-rich supermatrix approach on one-third of the extant species diversity calibrated with 23 macrofossils and two secondary calibration points. Our results corroborate previous studies and also suggest several new but poorly supported relationships. Newly suggested relationships are: (1) holoparasitic Mitrastemonaceae is sister to Lecythidaceae, (2) the clade formed by Mitrastemonaceae + Lecythidaceae is sister to Ericales excluding balsaminoids, (3) Theaceae is sister to the styracoids + sarracenioids + ericoids, and (4) subfamilial relationships with Ericaceae suggest that Arbutoideae is sister to Monotropoideae and Pyroloideae is sister to all subfamilies excluding Arbutoideae, Enkianthoideae, and Monotropoideae. Our results indicate Ericales began to diversify 110 Mya, within Indo-Malaysia and the Neotropics, with exchange between the two areas and expansion out of Indo-Malaysia becoming an important area in shaping the extant diversity of many families. Rapid cladogenesis occurred along the backbone of the order between 104 and 106 Mya. Jump dispersal is important within the order in the last 30 My, but vicariance is the most important cladogenetic driver of disjunctions at deeper levels of the phylogeny. We detect between 69 and 81 shifts in speciation rate throughout the order, the vast majority of which occurred within the last 30 My. We propose that range shifting may be responsible for older shifts in speciation rate, but more recent shifts may be better explained by morphological innovation.