The Artefactual Branch Effect and Phylogenetic Conflict: Species Delimitation with Gene Flow in Mangrove Pit Vipers (Trimeresurus purpureomaculatus-erythrurus Complex).

Mangrove pit vipers of the Trimeresurus purpureomaculatus-erythrurus complex are the only species of viper known to naturally inhabit mangroves. Despite serving integral ecological functions in mangrove ecosystems, the evolutionary history, distribution, and species boundaries of mangrove pit vipers remain poorly understood, partly due to overlapping distributions, confusing phenotypic variations, and the lack of focused studies. Here, we present the first genomic study on mangrove pit vipers and introduce a robust hypothesis-driven species delimitation framework that considers gene flow and phylogenetic uncertainty in conjunction with a novel application of a new class of speciation-based delimitation model implemented through the program Delineate. Our results showed that gene flow produced phylogenetic conflict in our focal species and substantiates the artefactual branch effect where highly admixed populations appear as divergent nonmonophyletic lineages arranged in a stepwise manner at the basal position of clades. Despite the confounding effects of gene flow, we were able to obtain unequivocal support for the recognition of a new species based on the intersection and congruence of multiple lines of evidence. This study demonstrates that an integrative hypothesis-driven approach predicated on the consideration of multiple plausible evolutionary histories, population structure/differentiation, gene flow, and the implementation of a speciation-based delimitation model can effectively delimit species in the presence of gene flow and phylogenetic conflict.

A comprehensive molecular phylogeny of the brachyuran crab superfamily Xanthoidea provides novel insights into its systematics and evolutionary history.

Maximum likelihood and Bayesian phylogenies for the brachyuran crab superfamily Xanthoidea were estimated based on three mitochondrial and four nuclear genes to infer phylogenetic relationships and inform taxonomy. Habitat data was then used in conjunction with several diversification rates analyses (BAMM, BiSSE, HiSSE, and FiSSE) to test evolutionary hypotheses regarding the diversification of xanthoid crabs. The phylogenies presented are the most comprehensive to date in terms of global diversity as they include all four constituent families (Xanthidae, Panopeidae, Pseudorhombilidae, and Linnaeoxanthidae) spanning all oceans in which xanthoid crabs occur. Six Xanthoidea families are recognised. Panopeidae and Xanthidae sensu stricto are the two largest family-level clades, which are reciprocally monophyletic. Pseudorhombilidae is nested within and is here treated as a subfamily of Panopeidae. Former subfamilies or tribes of Xanthidae sensu lato are basally positioned clades in Xanthoidea and are here assigned family-level ranks: Garthiellidae, Linnaeoxanthidae, Antrocarcinidae, and Nanocassiopidae. The genera Linnaeoxantho and Melybia were recovered in separate clades with Linnaeoxantho being sister to the family Antrocarcinidae, while Melybia was recovered within the family Panopeidae. The existing subfamily classification of Xanthidae and Panopeidae is drastically restructured with 20 xanthid and four panopeid subfamilies provisionally recognised. Diversification-time analyses inferred the origin of Xanthoidea and Garthiellidae in the Eocene, while the other families originated during the Oligocene. The majority of genus- and species-level diversification took place during the Miocene. Ancestral state reconstruction based on depth of occurrence (shallow vs. deep water) shows some ambiguity for the most recent common ancestor of Xanthoidea and Nanocassiopidae. The most recent common ancestors of Antrocarcinidae and Panopeidae were likely deep-water species, while those of Garthiellidae and Xanthidae were probably shallow-water species. Several shifts in net diversification rates were detected but they were not associated with depth-related habitat transitions.

Gene Flow Increases Phylogenetic Structure and Inflates Cryptic Species Estimations: A Case Study on Widespread Philippine Puddle Frogs (Occidozyga laevis).

In cryptic amphibian complexes, there is a growing trend to equate high levels of genetic structure with hidden cryptic species diversity. Typically, phylogenetic structure and distance-based approaches are used to demonstrate the distinctness of clades and justify the recognition of new cryptic species. However, this approach does not account for gene flow, spatial, and environmental processes that can obfuscate phylogenetic inference and bias species delimitation. As a case study, we sequenced genome-wide exons and introns to evince the processes that underlie the diversification of Philippine Puddle Frogs-a group that is widespread, phenotypically conserved, and exhibits high levels of geographically based genetic structure. We showed that widely adopted tree- and distance-based approaches inferred up to 20 species, compared to genomic analyses that inferred an optimal number of five distinct genetic groups. Using a suite of clustering, admixture, and phylogenetic network analyses, we demonstrate extensive admixture among the five groups and elucidate two specific ways in which gene flow can cause overestimations of species diversity: 1) admixed populations can be inferred as distinct lineages characterized by long branches in phylograms; and 2) admixed lineages can appear to be genetically divergent, even from their parental populations when simple measures of genetic distance are used. We demonstrate that the relationship between mitochondrial and genome-wide nuclear $p$-distances is decoupled in admixed clades, leading to erroneous estimates of genetic distances and, consequently, species diversity. Additionally, genetic distance was also biased by spatial and environmental processes. Overall, we showed that high levels of genetic diversity in Philippine Puddle Frogs predominantly comprise metapopulation lineages that arose through complex patterns of admixture, isolation-by-distance, and isolation-by-environment as opposed to species divergence. Our findings suggest that speciation may not be the major process underlying the high levels of hidden diversity observed in many taxonomic groups and that widely adopted tree- and distance-based methods overestimate species diversity in the presence of gene flow. [Cryptic species; gene flow; introgression; isolation-by-distance; isolation-by-environment; phylogenetic network; species delimitation.].

Target-capture phylogenomics provide insights on gene and species tree discordances in Old World treefrogs (Anura: Rhacophoridae).

Genome-scale data have greatly facilitated the resolution of recalcitrant nodes that Sanger-based datasets have been unable to resolve. However, phylogenomic studies continue to use traditional methods such as bootstrapping to estimate branch support; and high bootstrap values are still interpreted as providing strong support for the correct topology. Furthermore, relatively little attention has been given to assessing discordances between gene and species trees, and the underlying processes that produce phylogenetic conflict. We generated novel genomic datasets to characterize and determine the causes of discordance in Old World treefrogs (Family: Rhacophoridae)-a group that is fraught with conflicting and poorly supported topologies among major clades. Additionally, a suite of data filtering strategies and analytical methods were applied to assess their impact on phylogenetic inference. We showed that incomplete lineage sorting was detected at all nodes that exhibited high levels of discordance. Those nodes were also associated with extremely short internal branches. We also clearly demonstrate that bootstrap values do not reflect uncertainty or confidence for the correct topology and, hence, should not be used as a measure of branch support in phylogenomic datasets. Overall, we showed that phylogenetic discordances in Old World treefrogs resulted from incomplete lineage sorting and that species tree inference can be improved using a multi-faceted, total-evidence approach, which uses the most amount of data and considers results from different analytical methods and datasets.

Larger, unfiltered datasets are more effective at resolving phylogenetic conflict: Introns, exons, and UCEs resolve ambiguities in Golden-backed frogs (Anura: Ranidae; genus Hylarana).

Using FrogCap, a recently-developed sequence-capture protocol, we obtained >12,000 highly informative exons, introns, and ultraconserved elements (UCEs), which we used to illustrate variation in evolutionary histories of these classes of markers, and to resolve long-standing systematic problems in Southeast Asian Golden-backed frogs of the genus-complex Hylarana. We also performed a comprehensive suite of analyses to assess the relative performance of different genetic markers, data filtering strategies, tree inference methods, and different measures of branch support. To reduce gene tree estimation error, we filtered the data using different thresholds of taxon completeness (missing data) and parsimony informative sites (PIS). We then estimated species trees using concatenated datasets and Maximum Likelihood (IQ-TREE) in addition to summary (ASTRAL-III), distance-based (ASTRID), and site-based (SVDQuartets) multispecies coalescent methods. Topological congruence and branch support were examined using traditional bootstrap, local posterior probabilities, gene concordance factors, quartet frequencies, and quartet scores. Our results did not yield a single concordant topology. Instead, introns, exons, and UCEs clearly possessed different phylogenetic signals, resulting in conflicting, yet strongly-supported phylogenetic estimates. However, a combined analysis comprising the most informative introns, exons, and UCEs converged on a similar topology across all analyses, with the exception of SVDQuartets. Bootstrap values were consistently high despite high levels of incongruence and high proportions of gene trees supporting conflicting topologies. Although low bootstrap values did indicate low heuristic support, high bootstrap support did not necessarily reflect congruence or support for the correct topology. This study reiterates findings of some previous studies, which demonstrated that traditional bootstrap values can produce positively misleading measures of support in large phylogenomic datasets. We also showed a remarkably strong positive relationship between branch length and topological congruence across all datasets, implying that very short internodes remain a challenge to resolve, even with orders of magnitude more data than ever before. Overall, our results demonstrate that more data from unfiltered or combined datasets produced superior results. Although data filtering reduced gene tree incongruence, decreased amounts of data also biased phylogenetic estimation. A point of diminishing returns was evident, at which higher congruence (from more stringent filtering) at the expense of amount of data led to topological error as assessed by comparison to more complete datasets across different genomic markers. Additionally, we showed that applying a parameter-rich model to a partitioned analysis of concatenated data produces better results compared to unpartitioned, or even partitioned analysis using model selection. Despite some lingering uncertainties, a combined analysis of our genomic data and sequences supplemented from GenBank (on the basis of a few gene regions) revealed highly supported novel systematic arrangements. Based on these new findings, we transfer Amnirana nicobariensis into the genus Indosylvirana; and I. milleti and Hylarana celebensis to the genus Papurana. We also provisionally place H. attigua in the genus Papurana pending verification from positively identified (voucher substantiated) samples.

Multilocus phylogeny of Bornean Bent-Toed geckos (Gekkonidae: Cyrtodactylus) reveals hidden diversity, taxonomic disarray, and novel biogeographic patterns.

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.

To split or not to split? Multilocus phylogeny and molecular species delimitation of southeast Asian toads (family: Bufonidae).

BACKGROUND: Recent studies have demonstrated that Bayesian species delimitation based on the multispecies coalescent model can produce inaccurate results by misinterpreting population splits as species divergences. An approach based on the genealogical divergence index (gdi) was shown to be a viable alternative, especially for delimiting allopatric populations where gene flow is low. We implemented these analyses to assess species boundaries in Southeast Asian toads, a group that is understudied and characterized by numerous unresolved species complexes. RESULTS: Multilocus phylogenetic analyses showed that deep evolutionary relationships including the genera Sigalegalephrynus, Ghatophryne, Parapelophryne, Leptophryne, Pseudobufo, Rentapia, and Phrynoides remain unresolved. Comparison of genetic divergences revealed that intraspecific divergences among allopatric populations of Pelophyrne signata (Borneo vs. Peninsular Malaysia), Ingerophrynus parvus (Peninsular Malaysia vs. Myanmar), and Leptophryne borbonica (Peninsular Malaysia, Java, Borneo, and Sumatra) are consistent with interspecific divergences of other Southeast Asian bufonid taxa. Conversely, interspecific divergences between Pelophryne guentheri/P. api, Ansonia latiffi/A. leptopus, and I. gollum/I. divergens were low (< 3%) and consistent with intraspecific divergences of other closely related taxa. The BPP analysis produced variable results depending on prior settings and priors estimated from empirical data produced the best results that were also congruent with the gdi analysis. CONCLUSIONS: This study showed that the evolutionary history of Southeast Asian toads is difficult to resolve and numerous relationships remain ambiguous. Although some results from the species delimitation analyses were inconclusive, they were nevertheless efficacious at identifying potential new species and taxonomic incompatibilities for future in-depth investigation. We also demonstrated the sensitivity of BPP to different priors and that careful selection priors based on empirical data can greatly improve the analysis. Finally, the gdi can be a robust tool to complement other species delimitation methods.

Comprehensive multi-locus phylogeny of Old World tree frogs (Anura: Rhacophoridae) reveals taxonomic uncertainties and potential cases of over- and underestimation of species diversity.

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.

Species delimitation with gene flow: A methodological comparison and population genomics approach to elucidate cryptic species boundaries in Malaysian Torrent Frogs.

Accurately delimiting species boundaries is a nontrivial undertaking that can have significant effects on downstream inferences. We compared the efficacy of commonly used species delimitation methods (SDMs) and a population genomics approach based on genomewide single-nucleotide polymorphisms (SNPs) to assess lineage separation in the Malaysian Torrent Frog Complex currently recognized as a single species (Amolops larutensis). First, we used morphological, mitochondrial DNA and genomewide SNPs to identify putative species boundaries by implementing noncoalescent and coalescent-based SDMs (mPTP, iBPP, BFD*). We then tested the validity of putative boundaries by estimating spatiotemporal gene flow (fastsimcoal2, ABBA-BABA) to assess the extent of genetic isolation among putative species. Our results show that the A. larutensis complex runs the gamut of the speciation continuum from highly divergent, genetically isolated lineages (mean Fst  = 0.9) to differentiating populations involving recent gene flow (mean Fst  = 0.05; Nm  > 5). As expected, SDMs were effective at delimiting divergent lineages in the absence of gene flow but overestimated species in the presence of marked population structure and gene flow. However, using a population genomics approach and the concept of species as separately evolving metapopulation lineages as the only necessary property of a species, we were able to objectively elucidate cryptic species boundaries in the presence of past and present gene flow. This study does not discount the utility of SDMs but highlights the danger of violating model assumptions and the importance of carefully considering methods that appropriately fit the diversification history of a particular system.

Did true frogs 'dispersify'?

The interplay between range expansion and concomitant diversification is of fundamental interest to evolutionary biologists, particularly when linked to intercontinental dispersal and/or large scale extinctions. The evolutionary history of true frogs has been characterized by circumglobal range expansion. As a lineage that survived the Eocene-Oligocene extinction event (EOEE), the group provides an ideal system to test the prediction that range expansion triggers increased net diversification. We constructed the most densely sampled, time-calibrated phylogeny to date in order to: (i) characterize tempo and patterns of diversification; (ii) assess the impact of the EOEE; and (iii) test the hypothesis that range expansion was followed by increased net diversification. We show that late Eocene colonization of novel biogeographic regions was not affected by the EOEE and surprisingly, global expansion was not followed by increased net diversification. On the contrary, the diversification rate declined or did not shift following geographical expansion. Thus, the diversification history of true frogs contradicts the prevailing expectation that amphibian net diversification accelerated towards the present or increased following range expansion. Rather, our results demonstrate that despite their dynamic biogeographic history, true frogs diversified at a relatively constantly rate, even as they colonized the major land masses of Earth.

A new species of upland Stream Toad of the genus Ansonia Stoliczka, 1870 (Anura: Bufonidae) from northeastern Peninsular Malaysia.

A new species of Ansonia is described based on genetic and morphological differentiation. Ansonia lumut sp. nov. is most closely related to three other Peninsular Malaysian species, A. penangensis, A. malayana, and A. jeetsukumarani but differs from these and other congeners by at least 6.9% sequence divergence at the 12S, 16S rRNA and t-RNA-val genes and the following combination of morphological characters: (1) SVL 21.0-23.6 mm in males, 27.7-31.6 mm in females; (2) first finger shorter than second; (3) interorbital and tarsal ridges absent; (4) light interscapular spot absent; (5) presence of large, yellow rictal tubercle; (6) dorsum black with greenish-yellow reticulations; (7) flanks with small yellow spots; (8) fore and hind limbs with yellow cross-bars; and (9) venter light gray with fine, white spotting.

Next-generation bioinformatics: An ultrafast and user-friendly tool for phylogenomic data exploration.

With increasingly large genomic datasets, even routine bioinformatic tasks can be arduous, computationally demanding, and time-consuming. Additionally, most bioinformatic programs do not have a graphical user interface (GUI) and thus, require users to be minimally competent in command-line. These impediments present significant economic and technological barriers for practitioners who do not have access to advanced computational resources and support. In this issue of Molecular Ecology Resources, Handika and Esselstyn developed an ultrafast and memory-efficient bioinformatic tool, SEGUL, that performs common manipulations and calculations of summary statistics on phylogenomic datasets. SEGUL has two main features that distinguish it from other bioinformatic programs: (1) it is based on the recently emergent, high-performance programming language Rust, and (2) it has a GUI written using Flutter, a cross-platform programming framework that also supports mobile operating systems (mobile iOS, iPadOS and Android). By leveraging and combining the power of Rust and Flutter, SEGUL achieves significantly faster computation times and lower memory usage across different platforms and CPU architectures compared to similar programs. The unique and innovative features of SEGUL pave the way for a new era of bioinformatics that can be more energy-efficient, inclusive, and available to a broader swathe of users.

A new species of karst-adapted Cnemaspis Strauch, 1887 (Squamata: Gekkonidae) from a threatened karst region in Pahang, Peninsular Malaysia.

A new species of karst-adapted gekkonid lizard of the genus Cnemaspis Strauch is described from Gua Gunting and Gua Goyang in a karst region of Merapoh, Pahang, Peninsular Malaysia whose unique limestone formations are in immediate danger of being quarried. The new species differs from all other species of Cnemaspis based on its unique suite of morphological and color pattern characters. Its discovery underscores the unique biodiversity endemic to karst regions and adds to a growing list of karst-adapted reptiles from Peninsular Malaysia. We posit that new karst-adapted species endemic to limestone forests will continue to be discovered and these regions will harbor a significant percentage of Peninsular Malaysia's biodiversity and thusly should be conserved rather than quarried.

Scratching the surface: a new species of Bent-toed gecko (Squamata, Gekkonidae, Cyrtodactylus) from Timor-Leste of the darmandvillei group marks the potential for future discoveries.

A new species of limestone-dwelling Bent-toed gecko (genus Cyrtodactylus) is described from Nino Konis Santana National Park in the far-east region of Timor-Leste. Both genetic and morphological data strongly support the evolutionary distinctness of the new species, which we describe herein as Cyrtodactylussantana sp. nov. Phylogenetic analysis based on the ND2 mitochondrial gene inferred the new species as part of the C.darmandvillei group with close genetic affinities to C.batucolus, C.seribuatensis, C.petani, C.sadleiri, and two undescribed lineages from the Moluccas in Indonesia. The new species represents the first species of Cyrtodactylus identified at the species level from Timor-Leste and fills an important gap in our understanding of the biogeography and evolutionary history of Cyrtodactylus especially in the Wallacean region. Our results strongly suggest that the diversity of Cyrtodactylus in Wallacea is still underestimated and many more unnamed species remain to be described.

A new species of pit-viper from the Ayeyarwady and Yangon regions in Myanmar (Viperidae, Trimeresurus).

In a genomic study by Chan and colleagues, pit-vipers of the Trimeresuruserythrurus-purpureomaculatus complex from the Ayeyarwady and Yangon regions in Myanmar were demonstrated to be a distinct species based on robust population genetic and species delimitation analyses. Here, we provide morphological characterizations and a formal description of those populations as a new species. The new species, Trimeresurusayeyarwadyensissp. nov., is most closely related to T.erythrurus and T.purpureomaculatus and shares morphological characteristics with both of those species. Some specimens of T.ayeyarwadyensissp. nov. have green dorsal coloration and no distinct dorsal blotches (a trait shared with T.erythrurus but not T.purpureomaculatus), while others have dark dorsal blotches (a trait shared with T.purpureomaculatus but not T.erythrurus). The distinct evolutionary trajectory of the new species, coupled with the lack of obvious morphological differentiation, represents a classic example of the cryptic nature of species commonly found in the Trimeresurus group of Asian pit-vipers and underscores the need for data-rich analyses to verify species' boundaries more broadly within this genus.

GroupStruct: An R Package for Allometric Size Correction.

The efficacy of an allometric growth model to correct for ontogenetic body size variation has been known for decades, yet this method remains relatively obscure and rarely applied. We optimize the implementation of this method through a newly developed and easy-to-use R package GroupStruct and further extend its application from intraspecific to interspecific datasets. Using empirical examples, we show that different size correction methods (i.e., ratios, residuals, and allometry) can result in vastly different conclusions. Our results demonstrate that choosing the appropriate size correction method is crucial as it can have significant impacts on downstream analyses and has the potential to alter biological interpretations.

Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians.

BACKGROUND: The 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universal CO1 barcode that is more commonly used in other animal groups. However, studies employ different primer combinations that target different lengths/regions of the 16S gene ranging from complete gene sequences (~ 1500 bp) to short fragments (~ 500 bp), the latter of which is the most ubiquitously used. Sequences of different lengths are often concatenated, compared, and/or jointly analyzed to infer phylogenetic relationships, estimate genetic divergence (p-distances), and justify the recognition of new species (species delimitation), making the 16S gene region, by far, the most influential molecular marker in amphibian systematics. Despite their ubiquitous and multifarious use, no studies have ever been conducted to evaluate the congruence and performance among the different fragment lengths. RESULTS: Using empirical data derived from both Sanger-based and genomic approaches, we show that full-length 16S sequences recover the most accurate phylogenetic relationships, highest branch support, lowest variation in genetic distances (pairwise p-distances), and best-scoring species delimitation partitions. In contrast, widely used short fragments produce inaccurate phylogenetic reconstructions, lower and more variable branch support, erratic genetic distances, and low-scoring species delimitation partitions, the numbers of which are vastly overestimated. The relatively poor performance of short 16S fragments is likely due to insufficient phylogenetic information content. CONCLUSIONS: Taken together, our results demonstrate that short 16S fragments are unable to match the efficacy achieved by full-length sequences in terms of topological accuracy, heuristic branch support, genetic divergences, and species delimitation partitions, and thus, phylogenetic and taxonomic inferences that are predicated on short 16S fragments should be interpreted with caution. However, short 16S fragments can still be useful for species identification, rapid assessments, or definitively coupling complex life stages in natural history studies and faunal inventories. While the full 16S sequence performs best, it requires the use of several primer pairs that increases cost, time, and effort. As a compromise, our results demonstrate that practitioners should utilize medium-length primers in favor of the short-fragment primers because they have the potential to markedly improve phylogenetic inference and species delimitation without additional cost.

A standardized and statistically defensible framework for quantitative morphological analyses in taxonomic studies.

Although body size correction and inferential statistics have been used in morphological studies for many decades, their applications are far from being ubiquitous. We performed a meta-analysis to quantify the extent of taxonomic papers that performed body size correction and implemented a statistical hypothesis testing framework during the analysis of morphological data. Our results indicate that in most papers, neither of these analyses were performed but instead, cursory comparisons of descriptive statistics were presented. With the development of numerous freely available and powerful statistical programs such as R, we find it prudent to outline a standardized and statistically defensible framework to enhance the workflow of morphological analyses in taxonomic studies. This 5-step approach can be applied to meristic and mensural data across a wide range of taxonomic groups. We include an easy-to-use companion R script to facilitate the implementation of this workflow. Our proposed framework is not rooted in phylogenetic or evolutionary theory and hence, should not be used in place of explicit species delimitation techniques. Nevertheless, it can be incorporated into a more robust integrative taxonomic framework and is particularly useful for identifying diagnostic characters for species diagnoses.

The odd one in: re-diagnosis and phylogenetic placement of the Assam Day Gecko, Cnemaspis assamensis Das amp; Sengupta 2000 (Squamata: Gekkonidae).

Herein we provide new information on Cnemaspis assamensis, the only species of Cnemaspis known from north-eastern India. Based on five new samples, morphological parameters are described. The species was found to have pre-cloacal and femoral pores, not accounted for in the original description. Genetic assessment of the species was made using a 914 bp fragment of the ND2 mitochondrial gene and the species was recovered as the sister taxon of the C. podihuna clade from Sri Lanka.

Molecular Systematics of the Firefly Genus Luciola (Coleoptera: Lampyridae: Luciolinae) with the Description of a New Species from Singapore.

The firefly genus Luciola sensu McDermott contains 282 species that are distributed across major parts of Asia, Europe, Africa, Australia, and the Pacific islands. Due to phenotypic similarities, species identification using external morphological characters can be unreliable for this group. Consequently, decades of piecemeal taxonomic treatments have resulted in numerous erroneous and contentious classifications. Furthermore, our understanding of the group's evolutionary history is limited due to the lack of a robust phylogenetic framework that has also impeded efforts to stabilize its taxonomy. Here, we constructed molecular phylogenies of Luciola and its allies based on combined mitogenomes and Cytochrome c oxidase subunit 1 (COX1) sequences including a newly sequenced mitogenome of an unidentified taxon from Singapore. Our results showed that this taxon represents a distinct and hitherto undescribed evolutionary lineage that forms a clade with L. filiformis from Japan and L. curtithorax from China. Additionally, the Singaporean lineage can be differentiated from other congeners through several external and internal diagnostic morphological characters, and is thus described herein as a new species. Our phylogeny also strongly supported the paraphyly of Luciola with regard to L. cruciata and L. owadai, which were inferred to be more closely related to the genus Aquatica as opposed to other members of Luciola sensu stricto. The genus Hotaria was inferred as a derived clade within Luciola (sister to L. italica), supporting its status as a subgenus of Luciola instead of a distinct genus. This is the first time since 1909 that a new species of luminous firefly has been discovered in Singapore, highlighting the need for continued biodiversity research, even in small, well-studied and highly developed countries, such as Singapore.