Species Delimitation Analyses Research Articles

Global species delimitation of the cosmopolitan marine littoral earthworm Pontodrilus litoralis (Grube, 1855)

The marine littoral earthworm Pontodrilus litoralis (Grube, 1855) is widely distributed and is reported as a single species. This study utilized an integrative taxonomic approach based upon morphological examination, phylogenetic reconstruction, and molecular species delimitation, to test whether the taxon is a single species or a species complex. For this, a total of 114 P. litoralis specimens collected from North America, Africa, Australia and Oceania, Europe and Asia were used. The phylogenetic analyses revealed deeply divergent mitochondrial lineages and a high level of genetic diversity among P. litoralis populations. Both single and multi-locus species delimitation analyses yielded several molecular operational taxonomic units. Therefore, due to the homogeneity of morphological characteristics, it is likely that the morphospecies P. litoralis is a complex of four or more cryptic species, suggesting that more sampling is required and that the population structure genetic data and gene flow need to be investigated.

Towards a Canary Islands barcode database for soil biodiversity: revealing cryptic and unrecorded mite species diversity within insular soils.

Soil arthropod diversity contributes to a high proportion of the total biodiversity on Earth. However, most soil arthropods are still undescribed, hindering our understanding of soil functioning and global biodiversity estimations. Inventorying soil arthropods using conventional taxonomical approaches is particularly difficult and costly due to the great species richness, abundance and local-scale heterogeneity of mesofauna communities and the poor taxonomic background knowledge of most lineages. To alleviate this situation, we have designed and implemented a molecular barcoding framework adapted to soil fauna. This pipeline includes different steps, starting with a morphology-based selection of specimens which are imaged. Then, DNA is extracted non-destructively. Both images and voucher specimens are used to assign a taxonomic identification, based on morphology that is further checked for consistency with molecular information. Using this procedure, we studied 239 specimens of mites from the Canary Islands including representatives of Mesostigmata, Sarcoptiformes and Trombidiformes, of which we recovered barcode sequences for 168 specimens that were morphologically identified to 49 species, with nine specimens that could only be identified at the genus or family levels. Multiple species delimitation analyses were run to compare molecular delimitations with morphological identifications, including ASAP, mlPTP, BINs and 3% and 8% genetic distance thresholds. Additionally, a species-level search was carried out at the Biodiversity Databank of the Canary Islands (BIOTA) to evaluate the number of species in our dataset that were not previously recorded in the archipelago. In parallel, a sequence-level search of our sequences was performed against BOLD Systems. Our results reveal that multiple morphologically identified species correspond to different molecular lineages, which points to significant levels of unknown cryptic diversity within the archipelago. In addition, we evidenced that multiple species in our dataset constituted new records for the Canary Islands fauna and that the information for these lineages within online genetic repositories is very incomplete. Our study represents the first systematic effort to catalogue the soil arthropod mesofauna of the Canary Islands and establishes the basis for the Canary Islands Soil Biodiversity barcode database. This resource will constitute a step forward in the knowledge of these arthropods in a region of special interest.

Taxonomy and distribution of three species of Barleeia (Mollusca, Gastropoda, Littorinimorpha, Barleeiidae) in the intertidal zone off the coast of China, with description of a new species, Barleeia qilu Han, Qi & Kong, n. sp.

ABSTRACT Barleeiids are common marine microgastropods worldwide. Three species of Barleeia (Barleeia annamitica, Barleeia qilu n. sp. and Barleeia dunedini) found in the intertidal zone off the coast of China are described in this study. Morphological comparisons and species delimitation analyses using the universal mitochondrial COI gene sequence showed the distinctiveness of three species. Barleeia qilu n. sp., differentiated by its pointed apex and sutural channelling, was identified as a new species following morphological observation and phylogenetic analysis. Barleeia dunedini, previously considered as Ansola angustata (Pilsbry, 1901), has emerged as a distinct species and is probably the most widely distributed barleeiid in China. Barleeia annamitica, which has a broader distribution than previously recorded, is reported in China for the first time. Additionally, we propose to reassign Ansola angustata to the genus Barleeia based on the phylogram. Morphological illustrations, diagnoses, descriptions, and their approximate distribution along the coast of China are provided for each species. This study improves our understanding of the species composition, distribution and diversity of Barleeia in the intertidal zone of China. urn: lsid: zoobank.org:pub:7EFA6806-3A9E-40EC-A639-5AF4DB8D5CC7

Delimitation of the widely distributed Palearctic Stenodema species (Hemiptera, Heteroptera, Miridae): insights from molecular and morphological data.

Species delimitation presents a significant challenge in biology, particularly in systematics. Here, an integrative approach is employed to assess the species boundaries of widely distributed Palearctic Stenodema species. Due to their diversity, wide distribution, and the absence of comprehensive morphological and molecular data for most species, revising Stenodema is both daunting and time-consuming. Our study focuses on detailed examinations of male and female genitalia, coupled with phylogenetic analyses based on two mitochondrial markers (cytochrome c oxidase subunit I and 16S rRNA) and species delimitation analyses. Eight species with wide distributions are reviewed, Stenodematrispinosa Reuter, 1904 is synonymized with S.pilosa (Jakovlev, 1889), and a lectotype for Stenodematuranica Reuter, 1904 is designated. Morphological and molecular data effectively distinguish all species, revealing distinct clades and relationships. Notably, S.calcarata and S.pilosa form a well-supported clade, while S.virens and S.turanica share a lineage with Nearctic species. Stenodemarubrinervis and S.sibirica are morphologically similar and form a distinct clade in all phylogenies. Species delimitation analyses confirm the separation of all studied species, and genetic distances suggest the potential existence of cryptic species within S.calcarata and S.pilosa. This study highlights the advantages of integrative taxonomy in delimiting species with intricate and relatively recent phylogeographic histories.

DNA barcode shows discordant cases among morphological and molecular species identification in Isbrueckerichthys (Siluriformes: Loricariidae)

Abstract Isbrueckerichthys is a genus of armored catfish (Siluriformes: Loricariidae) with five species in the lowlands from the Ribeira de Iguape basin and in the uplands of the Tibagi River basin. Despite the validation of the morphological species, molecular data to investigate gene flow and species delimitation have not been completed for all species. For this purpose, we compared sequences of COI region associated with barcoding molecular identification, aiming for a species delimitation analysis and generating population data of I. alipionis, I. epakmos, I. duseni, I. cf. duseni, I. saxicola, and I. calvus. The K2P genetic distance, molecular species delimitation analysis, and well-sustained branches in the phylogenetic tree validate I. alipionis, I. epakmos, and I. duseni, and suggest I. cf. duseni as a valid molecular operational taxonomic unit. However, no differences were detected between I. saxicola and I. calvus. The discordance between molecular and morphological species may be due to the recent dispersal of some Isbrueckerichthys representatives at the border between the Ribeira de Iguape and Tibagi basins. The isolation features of the mountainous region of Ribeira de Iguape basin and headwaters captures to uplands are presented to explain the dispersion and the cases of incipient speciation in Isbrueckerichthys lineages.

Species Delimitation and Exploration of Species Partitions with ASAP and LIMES.

DNA barcoding plays an important role in exploring undescribed biodiversity and is increasingly used to delimit lineages at the species level (see Chap. 4 by Miralles et al.). Although several approaches and programs have been developed to perform species delimitation from datasets of single-locus DNA sequences, such as DNA barcodes, most of these were not initially provided as user-friendly GUI-driven executables. In spite of their differences, most of these tools share the same goal, i.e., inferring de novo a partition of subsets, potentially each representing a distinct species. More recently, a proposed common exchange format for the resulting species partitions (SPART) has been implemented by several of these tools, paving the way toward developing an interoperable digital environment entirely dedicated to integrative and comparative species delimitation. In this chapter, we provide detailed protocols for the use of two bioinformatic tools, one for single locus molecular species delimitation (ASAP) and one for statistical comparison of species partitions resulting from any kind of species delimitation analyses (LIMES).

DdRAD Sequencing and DNA Barcoding.

Double-digest restriction site-associated DNA sequencing is a library preparation protocol that enables capturing variable sites across the genome including single-nucleotide polymorphisms (SNPs). These SNPs can be utilized to gain evolutionary insights into patterns observed in DNA barcodes, to infer population structure and phylogenies, to detect gene flow and introgression, and to perform species delimitation analyses. The protocol includes chemically shearing genomic DNA with restriction enzymes, unique tagging, size selection, and amplification of the resulting DNA fragments. Here we provide a detailed description of each step of the protocol, as well as information on essential equipment and common issues encountered during laboratory work.

Delimiting Species with Single-Locus DNA Sequences.

DNA sequences are increasingly used for large-scale biodiversity inventories. Because these genetic data avoid the time-consuming initial sorting of specimens based on their phenotypic attributes, they have been recently incorporated into taxonomic workflows for overlooked and diverse taxa. Major statistical developments have accompanied this new practice, and several models have been proposed to delimit species with single-locus DNA sequences. However, proposed approaches to date make different assumptions regarding taxon lineage history, leading to strong discordance whenever comparisons are made among methods. Distance-based methods, such as Automatic Barcode Gap Discovery (ABGD) and Assemble Species by Automatic Partitioning (ASAP), rely on the detection of a barcode gap (i.e., the lack of overlap in the distributions of intraspecific and interspecific genetic distances) and the associated threshold in genetic distances. Network-based methods, as exemplified by the REfined Single Linkage (RESL) algorithm for the generation of Barcode Index Numbers (BINs), use connectivity statistics to hierarchically cluster-related haplotypes into molecular operational taxonomic units (MOTUs) which serve as species proxies. Tree-based methods, including Poisson Tree Processes (PTP) and the General Mixed Yule Coalescent (GMYC), fit statistical models to phylogenetic trees by maximum likelihood or Bayesian frameworks.Multiple webservers and stand-alone versions of these methods are now available, complicating decision-making regarding the most appropriate approach to use for a given taxon of interest. For instance, tree-based methods require an initial phylogenetic reconstruction, and multiple options are now available for this purpose such as RAxML and BEAST. Across all examined species delimitation methods, judicious parameter setting is paramount, as different model parameterizations can lead to differing conclusions. The objective of this chapter is to guide users step-by-step through all the procedures involved for each of these methods, while aggregating all necessary information required to conduct these analyses. The "Materials" section details how to prepare and format input files, including options to align sequences and conduct tree reconstruction with Maximum Likelihood and Bayesian inference. The Methods section presents the procedure and options available to conduct species delimitation analyses, including distance-, network-, and tree-based models. Finally, limits and future developments are discussed in the Notes section. Most importantly, species delimitation methods discussed herein are categorized based on five indicators: reliability, availability, scalability, understandability, and usability, all of which are fundamental properties needed for any approach to gain unanimous adoption within the DNA barcoding community moving forward.

Mitogenomic phylogeny, biogeography, and cryptic divergence of the genus Silurus (Siluriformes: Siluridae).

The genus Silurus, an important group of catfish, exhibits heterogeneous distribution in Eurasian freshwater systems. This group includes economically important and endangered species, thereby attracting considerable scientific interest. Despite this interest, the lack of a comprehensive phylogenetic framework impedes our understanding of the mechanisms underlying the extensive diversity found within this genus. Herein, we analyzed 89 newly sequenced and 20 previously published mitochondrial genomes (mitogenomes) from 13 morphological species to reconstruct the phylogenetic relationships, biogeographic history, and species diversity of Silurus. Our phylogenetic reconstructions identified eight clades, supported by both maximum-likelihood and Bayesian inference. Sequence-based species delimitation analyses yielded multiple molecular operational taxonomic units (MOTUs) in several taxa, including the Silurus asotus complex (four MOTUs) and Silurus microdorsalis (two MOTUs), suggesting that species diversity is underestimated in the genus. A reconstructed time-calibrated tree of Silurus species provided an age estimate of the most recent common ancestor of approximately 37.61 million years ago (Ma), with divergences among clades within the genus occurring between 11.56 Ma and 29.44 Ma, and divergences among MOTUs within species occurring between 3.71 Ma and 11.56 Ma. Biogeographic reconstructions suggested that the ancestral area for the genus likely encompassed China and the Korean Peninsula, with multiple inferred dispersal events to Europe and Central and Western Asia between 21.78 Ma and 26.67 Ma and to Japan between 2.51 Ma and 18.42 Ma. Key factors such as the Eocene-Oligocene extinction event, onset and intensification of the monsoon system, and glacial cycles associated with sea-level fluctuations have likely played significant roles in shaping the evolutionary history of the genus Silurus.

High species richness in the lichen genus Peltigera (Ascomycota, Lecanoromycetes): 34 species in the dolichorhizoid and scabrosoid clades of section Polydactylon, including 24 new to science.

Applying molecular methods to fungi establishing lichenized associations with green algae or cyanobacteria has repeatedly revealed the existence of numerous phylogenetic taxa overlooked by classical taxonomic approaches. Here, we report taxonomical conclusions based on multiple species delimitation and validation analyses performed on an eight-locus dataset that includes world-wide representatives of the dolichorhizoid and scabrosoid clades in section Polydactylon of the genus Peltigera. Following the recommendations resulting from a consensus species delimitation approach and additional species validation analysis (BPP) performed in this study, we present a total of 25 species in the dolichorhizoid clade and nine in the scabrosoid clade, including respectively 18 and six species that are new to science and formally described. Additionally, one combination and three varieties (including two new to science) are proposed in the dolichorhizoid clade. The following 24 new species are described: P. appalachiensis, P. asiatica, P. borealis, P. borinquensis, P. chabanenkoae, P. clathrata, P. elixii, P. esslingeri, P. flabellae, P. gallowayi, P. hawaiiensis, P. holtanhartwigii, P. itatiaiae, P. hokkaidoensis, P. kukwae, P. massonii, P. mikado, P. nigriventris, P. orientalis, P. rangiferina, P. sipmanii, P. stanleyensis, P. vitikainenii and P. willdenowii; the following new varieties are introduced: P. kukwae var. phyllidiata and P. truculenta var. austroscabrosa; and the following new combination is introduced: P. hymenina var. dissecta. Each species from the dolichorhizoid and scabrosoid clades is morphologically and chemically described, illustrated, and characterised with ITS sequences. Identification keys are provided for the main biogeographic regions where species from the two clades occur. Morphological and chemical characters that are commonly used for species identification in the genus Peltigera cannot be applied to unambiguously recognise most molecularly circumscribed species, due to high variation of thalli formed by individuals within a fungal species, including the presence of distinct morphs in some cases, or low interspecific variation in others. The four commonly recognised morphospecies: P. dolichorhiza, P. neopolydactyla, P. pulverulenta and P. scabrosa in the dolichorhizoid and scabrosoid clades represent species complexes spread across multiple and often phylogenetically distantly related lineages. Geographic origin of specimens is often helpful for species recognition; however, ITS sequences are frequently required for a reliable identification. Citation: Magain N, Miadlikowska J, Goffinet B, et al. 2023. High species richness in the lichen genus Peltigera (Ascomycota, Lecanoromycetes): 34 species in the dolichorhizoid and scabrosoid clades of section Polydactylon, including 24 new to science. Persoonia 51: 1-88. doi: 10.3767/persoonia.2023.51.01.

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.

RAD‐seq data provide new insights into biogeography, diversity anomaly, and species delimitation in eastern Asian–North American disjunct clade Benthamidia of Cornus (Cornaceae)

AbstractThe big‐bracted dogwood clade Benthamidia of Cornus is a typical example of the well‐known eastern Asia (EA) and North America (NA) floristic disjunction, with greater species diversity in EA than in NA. The lineage provides an opportunity to explore factors contributing to the plant diversity unevenness between EA and NA and test hypotheses on the origin of disjunct distribution from a phylogenetic perspective. We generated RAD‐seq data, conducted phylogenomic and biogeographic analyses for the clade with sampling of all species (9) and subspecies (10) currently recognized in floras. We also assessed species delineation and calculated phylogenetic diversity to evaluate the diversity unevenness between EA and NA. Finally, we examined variation of diversification rates and ecological niches on the phylogeny to explore potential causes underlying the observed diversity pattern. Our results revealed phylogenetic relationships congruent with previous studies and suggested a trans‐Beringian ancestral distribution of the clade Benthamidia in the mid‐Oligocene, dispersal from Mexico to eastern United States in the mid‐Miocene, and early diversification of the EA clade in SW China. Our results also confirmed greater phylogenetic diversity and diversification rate of the EA clade. Species delimitation analysis suggested 17 species in the clade Benthamidia, including all recognized subspecies. By integrating the results of molecular data with morphology, we proposed to retain the subspecies without changing their ranks. Our data suggested increased diversification rate in EA as an intrinsic factor explaining the greater species diversity in the region driven mainly by biogeographic isolation and partially by niche divergence.

Phylogenomics and species delimitation of an abundant and little-studied Amazonian forest spiny rat

Species delimitation studies based on integrating different datasets such as genomic, morphometric, and cytogenetics data are rare in studies focused on Neotropical rodents. As a consequence, the evolutionary history of most of these genera remains poorly understood. Proechimys is a highly diverse and widely distributed genus of Neotropical spiny rats with unique traits like multiple sympatry, micro-habitat segregation, and fuzzy species limits. Here, we applied RAD-Seq to infer the phylogenetic relationships, estimate the species boundaries, and estimate the divergence times for Proechimys, one of the most common and least studied small mammals in the Amazon. We tested whether inferred lineages in the phylogenetic trees could be considered distinct species based on the genomic dataset and morphometric data. Analyses revealed the genus is not monophyletic, with Proechimys hoplomyoides sister to a group of Hoplomys gymnurus + all other Proechimys species, contesting the generic status of Hoplomys. There are five main clades in Proechimys stricto sensu (excluding H. gymnurus and P. hoplomyoides). Species delimitation analyses supported 25 species within the genus Proechimys. The five main clades in Proechimys stricto sensu also showed similar ages for their origins, and two rapid diversification events were identified in the Early Pliocene and in the Early Pleistocene. Most cases of sympatry in Proechimys occur among species from the different main clades, and although Proechimys is an inhabitant of the Amazon, three species occupied the Cerrado biome during the Pleistocene. We could associate available nominal taxon, cytogenetics information, and DNA sequences in Genbank to most of the 25 species we hypothesized from our delimitation analyses. Based on our analyses, we estimate that eight forms represent putative new species that need a taxonomic revision.

Species delimitation and phylogenetic analyses of a New Guinean frog genus (Microhylidae: Hylophorbus) reveal many undescribed species and a complex diversification history driven by late Miocene events

Abstract New Guinea is the largest tropical island in the world and hosts immense endemic biodiversity. However, our understanding of how the gradual emergence of the terrestrial ecosystems of the island over the last 40 Myr has generated this biological richness is hampered by poorly documented species diversity and distributions. Here, we address both these issues through an integrative taxonomy and biogeographical approach using Hylophorbus, a New Guinea-endemic genus of frogs with 12 recognized species. We delimited candidate species by integrating mitochondrial DNA, nuclear DNA, and bioacoustics, then investigated their evolutionary history. Our results suggest that the current taxonomy of the genus misses true species diversity by ≥3.5-fold. Nevertheless, most candidate species (27) remain unconfirmed because of missing data, whereas five were identified unambiguously as undescribed (we describe three of these formally). Time-calibrated phylogenetic analyses suggest that Hylophorbus diversification began ~9 Mya in the northern or eastern portion of New Guinea. It would appear that lineages dispersed to new terrestrial habitats in the west, notably uplifted by the central range orogeny, until eventually reaching the Bird’s Head during the Mio-Pliocene (7–5 Mya). Conversely, a past barrier appears to have prevented north–south dispersal. These data suggest that new habitat availability has primarily driven the diversification of Hylophorbus.

Molecular population genetics and phylogeographic studies of Ligia exotica and Ligia cinerascens in East Asia

IntroductionSea slater, in the genus Ligia, is widespread in rocky shore habitats, and the taxon is easily isolated due to its limited dispersal capacity. Therefore, most Ligia species exhibit an allopatric distribution, but Ligia exotica and L. cinerascens exhibit an overlapping distribution distribution in East Asia. Previous studies on both species have confirmed the existence of highly divergent lineages based on 16S rRNA.MethodsIn the present study, 282 Ligia individuals were collected at ten, three, and three sites in South Korea, Japan, and Vietnam, respectively, and 41 haplotypes were observed based on 16S rRNA.Results and discussionThe results of phylogeny, phylogenetic network, and TCS network, Principal Coordinates Analysis, and four Molecular Species Delimitation Analyses revealed that six genetic lineages including L. cinerascens, Lineages N and S of L. exotica, Ligia sp. 1, sp.2 and sp.3 were present. The three genetic lineages, including L. cinerascens, Lineage N of L. exotica, and Lineage S of L. exotica, were also identified in the phylogeny based on a nuclear gene of the sodium–potassium ATPase α-subunit (Nak). Phylogeographic analysis revealed that L. cinerascens and Lineage N of L. exotica were distributed overlappingly in South Korea, Japan, and the northern region of China. Generally, the two lineages of L. exotica were distributed allopatrically, which was more evident along the coastline of mainland China than that of Japan. The results of time-calibrated phylogeny suggested that the speciation events of Ligia species might be associated with Japanese mainland formation from Oligocene to Miocene (approximately 30-5 million years ago, Mya). The results of the present study provide insights that could facilitate the understanding of the evolutionary history of Ligia, tracking of geological processes, and evolutionary effects of palaeogeographical events at the population level.

Phylogenomics and systematics of botryllid ascidians, and implications for the evolution of allorecognition

Allorecognition, the ability of an organism to distinguish kin from non-kin, or self from non-self, has been studied extensively in a group of invertebrate chordates, the colonial ascidians called botryllids (Subphylum Tunicata, Class Ascidiacea, Family Styelidae). When two conspecific botryllid colonies come in contact, there are two potential outcomes to an allorecognition reaction: fusion or rejection. The rejection outcome of allorecognition varies by species, and has been classified by type (referred to as R-Type). R-Type is defined according to how far the fusion process progresses before the rejection begins, since the rejection reaction appears as an interference of the fusion process. Here, we map the evolution of R-Types onto an extended and robust phylogeny of the botryllids. In this study, we have reconstructed the largest phylogenomic tree of botryllids, including 97 samples and more than 40 different species, and mapped on it nine of the 13 species for which the R-Type is known. Based on the R-Type known in a single outgroup species (Symplegma reptans), we infer that at least R-Type B and E-like could be ancestral to the Botrylloides/Botryllus group. However, the application of ancestral character state reconstructions does not provide conclusive results since several clades show more than one equiparsimonious R-Type state. Notably, all R-Type A species are clustered together and certainly evolved later than other R-Types. Our phylogenomic tree has been built on 177 nuclear loci and nearly all clades are well supported. Moreover, our phylogenetic analyses also take into account the results of species delimitation analyses based on the mitochondrial COI gene and of careful morphological analyses of the samples. The implementation of this integrated taxonomic approach, combining morphological as well as nuclear and mitochondrial data, has allowed the description of six new species, and the identification of a number of putative unnamed taxa. Thus, our results also demonstrate the existence of an unexplored hidden diversity within botryllids.

Lampreys in California (Lampetra spp. and Entosphenus spp.): Mitochondrial phylogenetic analysis reveals previously unrecognized lamprey diversity

AbstractObjectiveProtecting biodiversity is important for preserving ecosystem functions and services, and understanding the diversity present in a system is necessary for effective conservation. Lampreys display extensive diversity in morphology and life history strategy. The extent of this diversity and the underlying genetic patterns have not yet been fully characterized. Uncertainty about species boundaries and operational taxonomic units hinders lamprey management and conservation. Limited data have been collected on California lampreys (Lampetra spp. and Entosphenus spp.), but evidence suggests widespread population decline across the west coast of North America. Study objectives were to identify which nominal species are present at study sites across northern and central California, determine whether current taxonomic classifications accurately and sufficiently describe lamprey diversity in California, and characterize the biogeographic distribution of genetically distinct lamprey lineages across the study area.MethodsTo achieve these objectives, this study utilized DNA barcoding, phylogenetic analysis, and species delimitation analysis. Lamprey individuals (N = 87) from 19 sites in the Sacramento–San Joaquin River basin, San Francisco Bay, and Klamath River basin were sequenced for the mitochondrial cytochrome b (cyt b) gene, and the data were combined with publicly available lamprey cyt b sequences for analysis.ResultResults showed relatively deep phylogenetic divergence between Lampetra and Entosphenus. Distinct and genetically divergent lineages were observed within Lampetra, while distinct but genetically similar lineages were observed within Entosphenus. This study revealed novel Lampetra lineages in the Napa River and Alameda Creek, and results suggest that the biogeographic distribution of lamprey diversity may follow patterns observed in other native fishes. Species delimitation results indicated that at least seven separate candidate species of Lampetra can be found in California, suggesting that California holds more species‐level diversity than the expected three nominal Lampetra species known to occur in the state.ConclusionThese results highlight the underestimated diversity of lampreys in California and the need for further assessment of taxonomic classifications and operational taxonomic unit designations of California lampreys.

Phylogenetic analyses reveal multiple new stem-boring Tetramesa taxa (Hymenoptera: Eurytomidae): implications for the biological control of invasive African grasses

Many native South African grass species have become invasive elsewhere in the world. The application of biological control to invasive grasses has been approached with trepidation in the past, primarily due to concerns of a perceived lack of host specific herbivores. This has changed in recent times, and grasses are now considered suitable candidates. The Tetramesa Walker genus (Hymenoptera: Eurytomidae) has been found to contain species that are largely host specific to a particular grass species, or complex of closely related congeners. Very little taxonomic work exists for Tetramesa in the southern hemisphere, and the lack of morphological variability between many Tetramesa species has made identification difficult. This limits the ability to assess the genus for potential biological control agents. Species delimitation analyses indicated 16 putative novel southern African Tetramesa taxa. Ten of these were putative Tetramesa associated with Eragrostis curvula (Schrad.) Nees and Sporobolus pyramidalis Beauv. and S. natalensis Steud., which are alien invasive weeds in Australia. Of these ten Tetramesa taxa, eight were only found on a single host plant, while two taxa were associated with multiple species in a single grass genus. The Tetramesa spp. on S. pyramidalis and S. africanus were deemed suitably host-specific to be used as biological control agents. Field host range data for the Tetramesa species on E. curvula revealed that the wasp may not be suitably host specific for use as a biological control agent. However, further host specificity testing on non-target native Australian species is required.

Integrated phylogenetic analyses reveal the evolutionary, biogeographic, and diversification history of Asian warty treefrog genus Theloderma (Anura, Rhacophoridae).

Asian warty treefrogs, genus Theloderma, are morphologically variable arboreal frogs endemic to Southeast Asia and Southern China. However, integrated systematic studies are lacking, and knowledge of the genus in terms of diversity, origin, and historical diversification remains limited. To address these knowledge gaps, we used three mitochondrial and five nuclear gene fragments to reconstruct the Theloderma phylogeny, estimate divergence times, and examine the biogeography of the genus. Phylogenetic and species delimitation analyses suggest that the genus Theloderma comprises three major clades corresponding to two subgenera and seven species groups, and mPTP identified at least 12 putative cryptic species, suggesting that species diversity has been underestimated. Biogeographic analyses indicated that most recent common ancestor of Theloderma originated in the Indochina Peninsula during the Middle Oligocene (ca. 27.77 Ma) and the splitting of Clade A to C occurred in the Late Oligocene (ca. 23.55-25.57 Ma). Current biogeographic patterns result from two distinct processes: insitu diversification in the Indochina Peninsula and dispersal in multiple areas, namely southward dispersal to the Malay Peninsula and Borneo, northeastward dispersal to Southern China, northward dispersal to the Himalayas, and dispersal from Southern China to the Indochina Peninsula. Ancestral character reconstruction suggests that the ancestor of Theloderma may have possessed a small body size, rough dorsal skin, and absence of vomerine teeth and hand webbing, and that these four characters have undergone multiple evolutions. Principal component analysis based on eight bioclimatic variables did not clearly distinguish the three major clades of Theloderma, suggesting that species in these clades may occupy similar climatic ecological niches. Our research highlights the importance of orogeny and paleoclimatic changes, in shaping amphibian biodiversity in mountain ecosystems.

Discerning structure versus speciation in phylogeographic analysis of Seepage Salamanders (Desmognathus aeneus) using demography, environment, geography, and phenotype.

Numerous mechanisms can drive speciation, including isolation by adaptation, distance, and environment. These forces can promote genetic and phenotypic differentiation of local populations, the formation of phylogeographic lineages, and ultimately, completed speciation. However, conceptually similar mechanisms may also result in stabilizing rather than diversifying selection, leading to lineage integration and the long-term persistence of population structure within genetically cohesive species. Processes that drive the formation and maintenance of geographic genetic diversity while facilitating high rates of migration and limiting phenotypic differentiation may thereby result in population genetic structure that is not accompanied by reproductive isolation. We suggest that this framework can be applied more broadly to address the classic dilemma of "structure" versus "species" when evaluating phylogeographic diversity, unifying population genetics, species delimitation, and the underlying study of speciation. We demonstrate one such instance in the Seepage Salamander (Desmognathus aeneus) from the southeastern United States. Recent studies estimated up to 6.3% mitochondrial divergence and four phylogenomic lineages with broad admixture across geographic hybrid zones, which could potentially represent distinct species supported by our species-delimitation analyses. However, while limited dispersal promotes substantial isolation by distance, microhabitat specificity appears to yield stabilizing selection on a single, uniform, ecologically mediated phenotype. As a result, climatic cycles promote recurrent contact between lineages and repeated instances of high migration through time. Subsequent hybridization is apparently not counteracted by adaptive differentiation limiting introgression, leaving a single unified species with deeply divergent phylogeographic lineages that nonetheless do not appear to represent incipient species.