Molecular Phylogenetics and Evolution

The genus Pyrrhura (Psittacidae: Arini) is one of the most diverse groups of Neotropical parrots. Its species are charismatic, widely kept as pets, and frequently bred outside their native ranges. Yet, nearly half are currently listed as threatened by the IUCN within their natural distributions. Conservation assessments and population estimates often depend on the validity of accepted taxonomic boundaries. However, despite previous systematic efforts, the evolutionary relationships among and within many Pyrrhura species remain poorly resolved, largely due to a recent and rapid radiation. Here, we generated whole-genome sequences for all currently recognized Pyrrhura species, including multiple intraspecific taxa, to reconstruct a robust nuclear phylogeny under the multi-species coalescent model, alongside the most comprehensive mitogenome-based phylogeny of the genus to date. Although both phylogenies supported the monophyly of most currently accepted species, we identified several instances of mito-nuclear discordance, particularly involving the placement of early-diverging lineages, which are best explained by incomplete lineage sorting and historical gene flow. Additionally, we detected three distinct captive lineages that do not cluster with any known wild populations, suggesting substantial overlooked genetic diversity in the worlds captive populations. Ancestral range reconstructions indicate multiple and relatively recent colonization events into the northern and central Andes, likely associated with the uplift of the Andes and the emergence of new ecological niches. Together, our results reveal a complex evolutionary history in Pyrrhura, shaped by rapid radiations, incomplete lineage sorting, and gene flow. We show that integrating nuclear and mitochondrial data with broad geographic and taxonomic sampling, including captive individuals, can uncover overlooked genetic diversity and help to resolve long-standing systematic uncertainties. Finally, we show that several topological discrepancies among previous studies can be attributed to differences in sampling strategies, particularly within the most polytypic Pyrrhura species.

The delineation of closely related species remains a persistent challenge in Zoantharia, where morphological plasticity and limited genetic differentiation complicate taxonomy. In this study, we investigated the phylogenetic relationship between the widely distributed sibling taxa Palythoa tuberculosa (Indo-Pacific) and Palythoa caribaeorum (Atlantic) using ultraconserved elements (UCEs) recovered from genome skimming. A dataset comprising 116 loci (35,699 bp) across 37 specimens from Brazil, the Red Sea, Okinawa, and New Caledonia was analysed using both concatenated maximum-likelihood and coalescent-based approaches. Phylogenetic reconstructions did not recover monophyletic relationships corresponding to either species or geographic origin, instead revealing intermixed lineages across the Indo-Pacific and Atlantic regions. Concordance factor analyses indicated low gene concordance and moderate site concordance, suggesting pervasive gene tree discordance rather than a lack of phylogenetic signal. These patterns are consistent with previous studies based on mitochondrial, nuclear, and reduced-representation datasets, indicating that increased marker resolution does not resolve species boundaries within this complex. The observed lack of differentiation may reflect ongoing or recent connectivity among populations, potentially facilitated by long-distance dispersal promoted by anthropogenic rafting or historical range expansion, biological invasion, or biological processes such as incomplete lineage sorting. The results support the hypothesis that P. tuberculosa and P. caribaeorum represent a species complex or a case of incipient speciation rather than fully distinct evolutionary lineages. These findings indicate that genome-scale data alone may be insufficient to resolve very recent divergences, supporting the need for integrative approaches to resolve complicated species boundaries in zoantharians.

The shallow waters of the Tropical Eastern Pacific (TEP) harbor a species-rich octocoral fauna, with seven genera and 124 octocoral species described to date for the region. Of these lineages, Pacifigorgia, with 35 species, is by far the most speciose and abundant shallow-water octocoral occurring in the region. The speciation mechanisms resulting in this remarkable diversity remain speculative, despite the extensive taxonomic and molecular systematic research conducted so far in the TEP. Using genome-wide SNP markers, we provide evidence for hybridization and extensive cryptic speciation in Pacifigorgia, suggesting that the genus diversity has been underestimated by traditional and molecular systematic research. Our study highlights the difficulties faced by both traditional taxonomy and single-marker based molecular approaches to characterize octocoral diversity and evolution, and the role genome-wide molecular studies coupled to morphological research play to advance our understanding of this group.

The transition from structured populations to distinct species often unfolds along a speciation continuum. However, empirically dissecting this continuum poses challenges for species delimitation, particularly in rapid radiations marked by recent divergence, incomplete lineage sorting, and gene flow. The species-rich Sinocyclocheilus cavefish radiation of Southwest China, an emerging evolutionary multi-species model system, shows a notable mismatch between its high morphological diversity and the limited divergence observed in commonly used mitochondrial DNA markers. This pattern suggests that true species richness may be underestimated. Yet, comprehensive genome-wide approaches to resolve species boundaries in this group are still lacking. To address this, we combine phylogenomics, coalescent-based species delimitation, population genetics, and historical biogeography, using both genome-wide RAD-seq and multi-locus Sanger data (including nuclear and mitochondrial DNA). Our phylogenomic analyses resolve major clades and reveal substantial cryptic diversity, well beyond what was detected by earlier markers. Species delimitation based on RAD-seq genomic data identifies several cryptic evolutionary lineages. We recover a dynamic divergence history shaped by isolation and episodic connectivity. Biogeographic reconstruction supports a mid-Miocene origin following a major vicariance event, with subsequent founder-event dispersals into subterranean habitats. This long history of fragmentation is further complicated by reticulation and ancestral polymorphism, and is reflected in present-day patterns of restricted gene flow across river valleys. These results highlight the utility of integrative genomic approaches in resolving species boundaries and uncovering the evolutionary processes that underlie high diversity in large and complex radiations.

The temperate rainforests of the Pacific Northwest of North America harbor many endemic taxa whose evolutionary histories have been shaped by major climatic and geologic events. The enigmatic taildropper slugs (genus Prophysaon) are one example, notable for their ability to autonomize their tails to escape predators. Despite extensive work uncovering the evolutionary history of individual lineages, relationships among the nine recognized species of Prophysaon remain poorly understood due to insufficient molecular data. To address this, we collected transcriptomes for six of the nine currently accepted species of Prophysaon. Using these data, we were able to resolve species relationships, calling into question the existing subgeneric classification based on morphology. We also detected undescribed phenotypic diversity within the P. andersonii--P. foliolatum species complex, with molecular data supporting the distinctness of two phenotypically distinct populations from Washington. Finally, our transcriptomic data suggest a moderate role of introgression in shaping the evolutionary history of Prophysaon. Here, we synonymize the subgenus Mimetarion with nominotypical Prophysaon. Future work should further investigate whether the undescribed diversity detected here represents species level differentiation.

The vinegar fly Drosophila melanogaster is a pivotal model for invertebrate development, genetics, physiology, neuroscience, and disease. The whole family Drosophilidae, which contains over 4000 species, offers a plethora of cases for comparative and evolutionary studies. Despite a long history of phylogenetic inference, many relationships remain unresolved among the groups and genera in the Drosophilidae. To clarify these relationships, we first developed a set of new genomic markers and assembled a multilocus data set of 17 genes from 704 species of Drosophilidae. We then inferred well-supported group and species trees for this family. Additionally, we were able to determine the phylogenetic position of some previously unplaced species. These results establish a new framework for investigating the evolution of traits in fruit flies, as well as valuable resources for systematics.

Taxonomic classification, phylogenetics, and evolutionary history of night monkeys (Aotus) remain subjects of ongoing debate, particularly regarding species boundaries and the complex interplay of drivers underlying Neotropical primate diversification, including the historical role of the Amazon River as a biogeographic divide, Andean uplift, and Pliocene-Pleistocene climatic fluctuations. This study refines our understanding of Aotus systematics by employing fecal metagenomics to generate complete mitochondrial genomes from strategically collected samples across the lower Amazon Basin. Phylogenetic analyses integrating Maximum Likelihood, Bayesian inference, and species delimitation frameworks revealed several corrections to previously proposed biogeographic ranges and clarified evolutionary relationships among taxa. Phylogenomic reconstruction based on complete mitogenomes supports a reorganized classification into three principal clades--northern, western, and southern-- originating in the Early Pliocene. Additionally, an expanded mitochondrial cytochrome c oxidase subunit I (COI) dataset provided greater resolution of haplogroups within multiple species, revealing fine-scale geographic structure corresponding to major river barriers. Divergence time estimates were consistent with earlier studies, indicating a most recent common ancestor of the family Aotidae at approximately 17.2 million years ago and rapid diversification within Aotus beginning around 4.5 million years ago. Collectively, these results refine species distributions, illuminate the evolutionary history of Aotus, and provide an updated phylogeographic framework with direct implications for taxonomy and conservation management across the genus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/686292v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@5f5d6forg.highwire.dtl.DTLVardef@ef4eb6org.highwire.dtl.DTLVardef@1189663org.highwire.dtl.DTLVardef@1393e24_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical illustrationC_FLOATNO Phylogenetic relationships among Aotus taxa inferred from combined complete mitochondrial genomes and single-gene COI sequences using Bayesian Inference (BI). Branches are colored according to biogeographically defined taxonomic groups. Both datasets corroborate the recovery of three major clades (i.e., northern, western, and southern) and reveal fine-scale haplogroup structure corresponding to major Amazonian river barriers. Posterior probabilities are indicated at key nodes; only values <0.99 are shown. This integrated analysis highlights concordance between full mitogenomic and single-gene datasets, clarifying species boundaries and evolutionary relationships across the genus. C_FIG HighlightsO_LIComplete Aotus mitochondrial genomes were reconstructed from fecal samples across Amazon. C_LIO_LIExpanded COI analyses revealed haplogroups structured by rivers and areas of endemism. C_LIO_LIMisidentified sequences were corrected, clarifying A. boliviensis and A. trivirgatus. C_LIO_LIBiogeographic ranges were corrected, showing reduced distributions with conservation impact. C_LI

Two decades ago, the new siluriform family Lacantuniidae was erected to accommodate its sole known extant representative, Lacantunia enigmatica, a morphologically aberrant catfish restricted to the Middle Usumacinta River basin along the Guatemala-Mexico border. While its discovery was unexpected, its proposed phylogenetic placement--nested within a large clade of exclusively African-endemic families rather than closely related to regionally sympatric North American or Neotropical lineages--was baffling. To test this counterintuitive phylogenetic and biogeographic hypothesis, we collected new specimens of L. enigmatica and sequenced, assembled, and annotated for the first time its complete mitochondrial genome. We then constructed the most taxonomically comprehensive mitochondrial data matrix of catfishes to date by including published mitochondrial genomes representing the majority of siluriform families. Using Bayesian co-estimation of phylogeny and divergence times, we inferred the evolutionary position of the puzzling L. enigmatica within a time-scaled global catfish phylogeny. Our results offer improved resolution and understanding of higher-level siluriform relationships and refine the timescale of catfish evolution. Crucially, our findings corroborate the hypothesis that L. enigmatica is sister to the African family Claroteidae and represents a relict lineage that originated in Africa during the Late Cretaceous (74.99 Ma; 95% HPD=68.58-81.39) and eventually culminated in Middle America. Our results therefore uphold the necessity of transoceanic dispersal--as opposed to Gondwanan vicariance--to explain this otherwise puzzling biogeographic pattern.

Lack of divergence in mitochondrial DNA between species with clear phenotypic differences may be the result of low resolution of markers, incomplete lineage sorting, introgression, or the interplay of various evolutionary mechanisms acting on different traits and genomic regions through time. Previous work revealed that the Andean hummingbirds Coeligena bonapartei and C. helianthea lack genetic divergence in the mitochondrial ND2 gene, which shows variation discordant with coloration phenotype but consistent with geography. We sequenced and analyzed complete mitochondrial genomes for C. b. bonapartei, C. b. consita, C. h. helianthea and C. h. tamai to assess whether patterns revealed by ND2 analyses hold when considering the entire mitogenome, and to shed light into the evolutionary history of these hummingbirds. We found very low genetic differentiation in mitogenomes among the four lineages of Coeligena, confirming patterns based on ND2 data. Estimates of genetic differentiation, phylogenies and haplotype network analyses of complete mitogenomes did not separate phenotypically distinct taxa, but were consistent with a previously described pattern of northern vs. southern divergence along the Cordillera Oriental of Colombia. Mitogenomes of C. b. bonapartei and C. h. helianthea are indistinguishable, suggesting incomplete lineage sorting or strong introgression. Mitogenomes of C. b. consita and C. h. tamai are slightly differentiated, but they are more similar to each other than either is to that of its respective nominate subspecies, a result also suggestive of mtDNA introgression despite distinct phenotypic differences. Our results indicate that various evolutionary mechanisms playing out over a complex biogeographic scenario in the Colombian Andes drove divergence in phenotypes and mitochondrial genomes of Coeligena hummingbirds, and lead to alternative hypotheses to be tested with whole-genome analyses.

The order Atheriniformes (silversides, rainbowfishes, and blue-eyes) is a globally distributed group of fishes with frequent evolutionary transitions between marine and freshwater ecosystems. However, understanding the tempo and mode of these transitions has been hampered by poor phylogenetic resolution and limited taxonomic sampling, particularly within the suborder Atherinoidei. We generated a phylogenomic dataset of 1,100 exon loci for 150 species to resolve interfamilial relationships and reconstruct the groups biogeographic history. We were also able to incorporate a large number of existing GenBank sequences, producing a phylogeny with 265 species sampled for at least some genetic data (67% of known species diversity). While the New World suborder Atherinopsidae is well-resolved, we found that the family Atherinidae is polyphyletic across all analyses. We propose a revised classification that restricts Atherinidae to the genus Atherina and recognizes Atherinomoridae and Craterocephalidae as separate families. Our biogeographic inferences using explicit geographic areas suggests more frequent marine-to-freshwater transitions than previously inferred with simplified binary (marine vs. freshwater) coding, and uncover habitat transitions where marine ancestors may have gone extinct. These results highlight how explicit geographic modeling can uncover marine ancestry erased by extinction, providing a robust phylogenetic framework for future evolutionary studies of Atheriniformes.

Macaws of the genus Ara comprise eight extant species distributed throughout the Neotropics. Among them, four have broad geographic ranges, yet little is known about the evolutionary history and demographic processes that shaped their genomic variation and present-day distributions. This is particularly relevant because, although these wide-ranging macaws are classified as Least Concern by the IUCN, many of their populations are declining due to habitat fragmentation, illegal trade, and climate change. Here, we used nuclear and mitochondrial genomic data to characterize the evolutionary relationships, population structure, genetic diversity, and demographic histories of three widely distributed species (A. ararauna, A. chloropterus, and A. severus) across their geographic distributions. We identified two main populations within Ara severus, and this species showed the highest heterozygosity levels among the three species. In A. ararauna and A. chloropterus, we observed four main genetic clusters corresponding to two populations in the Amazon rainforest biome and and two populations in the Cerrado savanna biome. Cerrado populations in both species exhibited markedly reduced heterozygosity and elevated inbreeding relative to Amazonian populations, consistent with smaller effective population sizes and increased isolation. Genome-wide scans suggested that genetic drift and divergent demographic histories played a predominant role in driving the strong differentiation between Amazon and Cerrado in these two species. Nevertheless, we detected two candidate genes, NALCN and RBBP6, with convergent selection signals across A. ararauna and A. chloropterus, suggesting possible local adaptation to the Cerrado biome.

AO_SCPLOWBSTRACTC_SCPLOWThis study reconstructs the phylogeny of an expansive set of Neotropical leaf beetles in the subfamily Chrysomelinae. From 33 species in the genus Platyphora Gistel, and an additional 37 species representing 16 beetle genera, five genes, three nuclear, and two mitochondrial, were sequenced and used to obtain a well-supported molecular phylogeny using both Bayesian and Maximum Likelihood. The subtribes Chrysomelina and Doryphorina (sensu Daccordi 1982) were monophyletic, while the genus Platyphora was polyphyletic. The genus Leptinotarsa Chevrolat is confirmed to be distinct from Stilodes Chevrolat. Host plant family was recorded for both adults and larvae using direct observations where possible. Ancestral host plant use was reconstructed using Bayesian trait analyses. A complicated history of host plant switches among a restricted set of plant families is revealed: In the paraphyletic Platyphora, one clade that includes Proseicela and Leptinotarsa had two switches from Asclepiadiodeae to Solanaceae, one switch to Moraceae, and one switch to Malpighiaceae, another Platyphora clade had switches between Asteraceae and Rauvolfioideae, and from Rauvolfioideae to Asclepiadiodeae, with other members of the same clade feeding on Boraginaceae and Convolvulaceae. All species included in the clade containing Tritaenia and Stilodes fed on Malpighiaceae, and all species included in the Cosmogramma and Calligrapha clade fed on Malvaceae.

Macromiidae is a widely distributed lineage of libelluloid dragonflies with a largely allopatric genus-level distribution across the Holarctic, Afrotropical, Australasian, and Indo-Malayan regions. Previous studies involving this family have been complicated by morphological convergence and limited phylogenetic sampling. Here, we present the most densely sampled phylogenetic framework for Macromiidae to date, using Anchored Hybrid Enrichment data from 62 of the 125 described species. Our sampling represents all four genera and major geographic regions, including Libelluloid and Cordulegastrid outgroups. Maximum likelihood recovered three major lineages: Epophthalmia, Phyllomacromia, and Macromia sensu lato, with Epophthalmia strongly supported as sister to Phyllomacromia. Didymops was not recovered as monophyletic and was placed within Macromia, although deeper relationships within the Macromia complex showed some gene tree discordance. We additionally scored seven male genitalic characters and reconstructed their evolution across a dated phylogeny. We revealed that these traits varied heavily in phylogenetic signal, with some characters supporting the major clades and others showing high degree of homoplasy. Fossil-calibrated divergence time estimation placed the crown origin of Macromiidae in the late Oligocene (24 Ma), with other major intrafamilial divergences concentrated in the Miocene. Historical biogeographic reconstructions consistently supported Afrotropical origins for Phyllomacromia, Indo-Malayan centered ancestry for Epophthalmia, and a multi-region history for Macromia + Didymops spanning Indo-Malayan, Australasian, and Nearctic regions. Habitat reconstructions favored lentic ancestry for Macromiidae, and diversification rate variation was best explained by trait-independent models rather than lentic/lotic habitat association.

Robber flies or assassin flies (Diptera: Asilidae) are a diverse family of venomous predators. The most recent classification organizes Asilidae into 14 subfamilies based on a comprehensive morphological phylogeny, but many of these have not been supported in a subsequent molecular study using traditional molecular markers. To address questions of monophyly in Asilidae, we leveraged the recently developed Diptera-wide UCE baitset to compile seven datasets comprising 151 robber flies and 146 - 2,508 loci, varying in the extent of missing data. We also studied the behavior of different nodal support metrics, as the non-parametric bootstrap is known to perform poorly with large genomic datasets. Our ML phylogeny was fully resolved and well-supported, but partially incongruent with the coalescent phylogeny. Further examination of the datasets suggested the possibility that GC bias had influenced gene tree inference and subsequent species tree analysis. The subfamilies Brachyrhopalinae, Dasypogoninae, Dioctriinae, Stenopogoninae, Tillobromatinae, Trigonomiminae, and Willistonininae were not recovered as monophyletic in either analysis, consistent with a previous molecular study. The inter-subfamily relationships are summarized as follows: Laphriinae and Dioctriinae (in part) are successively sister to the remaining subfamilies, which form two clades; the first consists of a grade of Stenopogoninae (in part), Willistonininae (in part), Bathypogoninae+Phellinae, Stichopogoninae, Leptogastrinae, Ommatiinae, and Asilinae; the second clade consists of a thoroughly paraphyletic assemblage of genera from Dioctriinae (in part), Trigonomiminae, Stenopogoninae (in part), Tillobromatinae, Brachyrhopalinae, and Dasypogoninae. We find that nodal support does not significantly vary with missing data. Furthermore, the bootstrap appears to overestimate nodal support, as has been reported from many recent studies. Gene concordance and site concordance factors seem to perform better, but may actually underestimate support. We instead recommend quartet concordance as a more appropriate estimator of nodal support. Our comprehensive phylogeny demonstrates that the higher classification of Asilidae is far from settled, and it will provide a much-needed foundation for a thorough revision of the subfamily classification.

Despite tropical psocids comprise ~60% of species diversity within the Psocidae (Insecta, Psocodea), previous studies on the Psocidae phylogeny have poorly sampled tropical species (<40% species in trees). Here we discuss the evolution and systematics of the Psocidae based on the most comprehensive species-level sampling of the Psocidae. We sequenced and inferred the phylogenetic position of 43 previously unsampled Neotropical species from COI, H3, WNT, 18S, 16S, and 12S. Based on our phylogenies we found that Neotropical psocids are generally not closely related to morphologically similar taxa in the Holarctic region. Consequently, the monophyletic status for the major groups within Psocidae (subfamilies and tribes) is recovered only when Holarctic groups are sampled (7-10 of 11 higher-level groups are monophyletic) but violated when Neotropical species are included in the dataset (1 of 11 higher-level groups are monophyletic). Leveraging the largest phylogeny of the Psocidae, our study pinpoints the downfalls of simply extending taxonomic knowledge from lineages of a certain area to inform diversity and evolution of lineages in other regions. HighlightsO_LITropical psocids comprise >60% of the extant family richness C_LIO_LIPrevious phylogenies have undersampled Tropical psocids C_LIO_LIHolarctic and Neotropical species are classified under the same morphological groups C_LIO_LIHolarctic and Neotropical generally correspond to evolutionarily distinct lineages C_LIO_LIPhylogenies based on Holarctic psocids poorly inform evolution in the Neotropics C_LI

The Formicoxenus genus-group comprises six genera within the tribe Crematogastrini. The group is well known for repeated evolution of social parasitism among closely related taxa and cold-adapted species with large distribution ranges in the Nearctic and Palearctic regions. Previous analyses based on nuclear markers (ultraconserved elements, UCEs) and mitochondrial genes suggest close relationship between Formicoxenus Mayr, 1855, Leptothorax Mayr, 1855 and Harpagoxenus Forel, 1893. However, scant sampling has limited phylogenetic assessment of these genera. Also, previous phylogeographic analyses of L. acervorum (Fabricius, 1793) have been limited to its West-Palearctic range of distribution, which has provided a narrow view on recolonization, population structure and existing refugia of the species. Here, we inferred the phylogenenetic history of genera within the Formicoxenus genus-group and reconstructed the phylogeography of L. acervorum with more extensive sampling. We employed four datasets consisting of whole genomes and sequences of the COI. The topologies of previous nuclear and our inferences based on mitochondrial genomes were overall congruent. Further, Formicoxenus may not be monophyletic. We found several monophyletic lineages that do not correspond to the current species described within Leptothorax, especially in the Nearctic region. We identified a monophyletic L. acervorum lineage that comprises both Nearctic and Palearctic locations. The most recent expansion within L. acervorum probably occurred within the last 0.5 Ma with isolated populations predating the Last Glacial Maximum (LGM), which are localized in at least two refugial areas (Pyrenean and Northern plateau) in the Iberian Peninsula. The patterns recovered suggest a shared glacial refugium in the Iberian Peninsula with cold-adapted trees that currently share high-altitude environments in this region.

Lamprologine cichlids are a diverse group of fishes distributed in Lake Tanganyika and the Congo River. Nine species of Lamprologus occur in the Congo River basin including the only blind cichlid Lamprologus lethops, but little is known about the natural history and evolution of this enigmatic species. To alleviate this knowledge gap, we characterized the complete mitochondrial genomes of L. lethops and its riverine congeners and provided a phylogenetic hypothesis based on these data. We recovered complete mitochondrial genomes from eleven specimens of eight species of Lamprologus. Mitogenomes were identical in the number and order of genes and similar in size (16,579-16,587 bp). In contrast to previous phylogenomic studies, riverine Lamprologus were recovered in two non-sister mitochondrial lineages that were more closely related to other genera of Lake Tanganyika lamprologines than to each other. In the first lineage, most relationships were not highly supported. In the second lineage, L. lethops was recovered as the sister species of L. markerti, L. mocquardi and L. tigripictilis. Interestingly, sequences from L. mocquardi were found in the two mitochondrial lineages. Our results hint at multiple events of past introgression and highlight the importance of increasing taxonomic and genomic sampling to study complex evolutionary histories.

Scolopendra Linnaeus, 1758 is a genus of centipede showing reasonably good morpho-molecular correlation. This often allows the acceptable species differentiation and phylogenetic positioning by just using the partial sequence of the cytochrome c oxidase (COI) gene. However, for biological, statistical, or technical reasons, several exceptions to that fact have been observed, which together with a lack of a solid criterion to differentiate species at the molecular level, make difficult the interpretation of some taxonomic and systematic outcomes. With the scope of providing a standardized system improving the molecular Scolopendra species delimitation and surpassing some issues related with the mtDNAs use, a total of 45 representative COI sequences belonging to 22 taxa are tested, but for the first time, performing distinct amino acids chains evolution analyses. To illustrate this, a general evaluation of the genus is firstly provided, while to exemplify some of the deeper analysis, the cases of S. paradoxa Domenech, 2018 and S. spinosissima Kraepelin, 1903 are then here re-explored using some alternative tools. As a result, the partial COI protein sequences analysis identified, at least, ten amino acid residues positions as useful for Scolopendra species differentiation, being generally five divergent residues enough to distinguish taxa ["the five divergent amino acid rule"]. When this premise wasnt fulfilled, the amino acid frequency, exclusivity, or electrochemical properties and especially the r substitution probability test, helped solve the cases. Among S. paradoxa and S. spinosissima five diverging residues were found, with one of them being exclusive to the entire genus for the former taxon. Also, the substitution probability (r) analysis showed strong positive selection for three of these five divergent amino acids. As exceptions, S. dawydoffi Kronmuller, 2012 has been found indistinguishable from S. multidens Newport, 1844 by this method, suggesting its eventual synonymy, while S. cataracta Siriwut, Edgecombe & Panha, 2016 showed an intraspecific maximum divergence of 5 amino acids. Finally and at nomenclatural level, the species name S. hainanum Kronmuller, 2012 is emended as S. hainanensis while the taxon S. mojiangica Zhang & Chi, is here declared nomen nudum, due its original description remains unavailable.

Ciliates are single-celled microbial eukaryotes that diverged from other eukaryotic lineages over a billion years ago. The extensive evolutionary timespan of ciliate has led to enormous genetic and phenotypic changes, contributing significantly to their high level of diversity. Recent analyses based on molecular data have revealed numerous cases of cryptic species complexes in different ciliate lineages, demonstrating the need for a robust approach to delimit species boundaries and elucidate phylogenetic relationships. Heterotrich ciliate species of the genus Spirostomum are abundant in freshwater and brackish environments and are commonly used as biological indicators for assessing water quality. However, some Spirostomum species are difficult to identify due to a lack of distinguishable morphological characteristics, and the existence of cryptic species in this genus remains largely unexplored. Previous phylogenetic studies have focused on only a few loci, namely the ribosomal RNA genes, alpha-tubulin, and mitochondrial CO1. In this study, we obtained single-cell transcriptome of 25 Spirostomum species populations (representing six morphospecies) sampled from South Korea and the USA, and used concatenation- and coalescent-based methods for species tree inference and delimitation. Phylogenomic analysis of 37 Spirostomum populations and 265 protein-coding genes provided a robustious insight into the evolutionary relationships among Spirostomum species and confirmed that species with moniliform and compact macronucleus each form a distinct monophyletic lineage. Furthermore, the multispecies coalescent (MSC) model suggests that there are at least nine cryptic species in the Spirostomum genus, three in S. minus, two in S. ambiguum, S. subtilis, and S. teres each. Overall, our fine sampling of closely related Spirostomum populations and wide scRNA-seq allowed us to demonstrate the hidden crypticity of species within the genus Spirostomum, and to resolve and provide much stronger support than hitherto to the phylogeny of this important ciliate genus.

The systematics of the family Dendrodorididae poses a challenge to evolutionary biologists, as their mitochondrial and nuclear markers provide contradictory phylogenetic signals. Nuclear pseudogenes or exogenous contamination are hypothesized to cause the molecular discordance. However, these hypotheses have not been tested. We used genomic data from seven Dendrodorididae species to investigate the evolution of this family. Two mitogenomes displayed a novel structural rearrangement in nudibranchs, involving the translocation of three collinear genes and five surrounding tRNAs. Additionally, we found numerous mitogenomic regions with non-synonymous mutations and multiple indels in both coding and ribosomal genes. Protein modeling resulted in similar structures, suggesting that functionality is conserved. Phylogenies using mitogenomic data confirmed a specific clade membership for the rearranged mitogenomes. The incorporation of nuclear data did not fully resolve the systematic relationships of Dendrodorididae, acknowledging the evolutionary complexity of this group. The present study provides novel evidence on sudden molecular changes in mitogenomes, and highlights the relevance of using genomic data to unveil rare evolutionary processes, which is critical for understanding evolution of neglected taxa.